SRPKIN-1: A Covalent SRPK1/2 Inhibitor that Potently Converts VEGF from Pro-angiogenic to Anti-angiogenic Isoform Author links open overlay panel

John M.Hatcher, Guowei Wu, Chuyue Zeng, JieZhu, Fan Meng, Sherrina Patel, Wenqiu Wang, Scott B.Ficarro, Alan L.Leggett, Chelsea E.Jarrod A.Marto, Kang Zhang, Jacky ChiKi Ngo, Xiang-Dong Fu, Tinghu Zhang, Nathanael S.Gray

Cell. Chem. Biol, 2018

https://doi.org/10.1016/j.chembiol.2018.01.013

The SRPK family of kinases regulates pre-mRNA splicing by phosphorylating serine/arginine (SR)-rich splicing factors, signals splicing control in response to extracellular stimuli, and contributes to tumorigenesis, suggesting that these splicing kinases are potential therapeutic targets. Here, we report the development of the first irreversible SRPK inhibitor, SRPKIN-1, which is also the first kinase inhibitor that forms a covalent bond with a tyrosine phenol group in the ATP-binding pocket. Kinome-wide profiling demonstrates its selectivity for SRPK1/2, and SRPKIN-1 attenuates SR protein phosphorylation at submicromolar concentrations. Vascular endothelial growth factor (VEGF) is a known target for SRPK-regulated splicing and, relative to the first-generation SRPK inhibitor SRPIN340 or small interfering RNA-mediated SRPK knockdown, SRPKIN-1 is more potent in converting the pro-angiogenic VEGF-A165a to the anti-angiogenic VEGF-A165b isoform and in blocking laser-induced neovascularization in a murine retinal model. These findings encourage further development of SRPK inhibitors for treatment of age-related macular degeneration.

2‑Formylpyridyl Ureas as Highly Selective Reversible-Covalent Inhibitors of Fibroblast Growth Factor Receptor 4

Thomas Knoepfel, Pascal Furet, Robert Mah, Nicole Buschmann, Catherine Leblanc, Sebastien Ripoche, Diana Graus-Porta, Markus Wartmann, Inga Galuba, and Robin A. Fairhurst

ACS Med. Chem. Lett., Article ASAP
DOI: 10.1021/acsmedchemlett.7b00485

 As part of a project to identify FGFR4 selective inhibitors, scaffold morphing of a 2-formylquinoline amide hit identified series of 2-formylpyridine ureas (2-FPUs) with improved potency and physicochemical properties. In particular, tetrahydronaphthyridine urea analogues with cellular activities below 30 nM have been identified. Consistent with the hypothesized reversible-covalent mechanism of inhibition, the 2-FPUs exhibited slow binding kinetics, and the aldehyde, as the putative electrophile, could be demonstrated to be a key structural element for activity.

L718Q mutant EGFR escapes covalent inhibition by stabilizing a non-reactive conformation of the lung cancer drug osimertinib

D. Callegari, K. E. Ranaghan, C. J. Woods, R. Minari, M. Tiseo, M. Mor, A. J. Mulholland, and A. Lodola

Chem. Sci., 2018
DOI: 10.1039/C7SC04761D

Osimertinib is a third-generation inhibitor approved for the treatment of non-small cell lung cancer. It overcomes resistance to first-generation inhibitors by incorporating an acrylamide group which alkylates Cys797 of EGFR T790M. The mutation of a residue in the P-loop (L718Q) was shown to cause resistance to osimertinib, but the molecular mechanism of this process is unknown. Here, we investigated the inhibitory process for EGFR T790M (susceptible to osimertinib) and EGFR T790M/L718Q (resistant to osimertinib), by modelling the chemical step (i.e., alkylation of Cys797) using QM/MM simulations and the recognition step by MD simulations coupled with free-energy calculations. The calculations indicate that L718Q has a negligible impact on both the activation energy for Cys797 alkylation and the free-energy of binding for the formation of the non-covalent complex. The results show that Gln718 affects the conformational space of the EGFR–osimertinib complex, stabilizing a conformation of acrylamide which prevents reaction with Cys797.

Targeting KRAS Mutant Cancers with a Covalent G12C-Specific Inhibitor

Matthew R. Janes, Jingchuan Zhang, Lian-Sheng Li, Rasmus Hansen, Ulf Peters, Xin Guo, Yuching Chen, Anjali Babbar, Sarah J. Firdaus, Levan Darjania, Jun Feng, Jeffrey H. Chen, Shuangwei Li, Shisheng Li, Yun O. Long, Carol Thach, Yuan Liu, Ata Zarieh, Tess Ely, Jeff M. Kucharski, Linda V. Kessler, Tao Wu, Ke Yu, Yi Wang, Yvonne Yao, Xiaohu Deng, Patrick P. Zarrinkar, Dirk Brehmer, Dashyant Dhanak, Matthew V. Lorenzi, Dana Hu-Lowe, Matthew P. Patricelli, Pingda Ren, Yi Liu

DOI: http://dx.doi.org/10.1016/j.cell.2018.01.006

KRASG12C was recently identified to be potentially druggable by allele-specific covalent targeting of Cys-12 in vicinity to an inducible allosteric switch II pocket (S-IIP). Success of this approach requires active cycling of KRASG12C between its active-GTP and inactive-GDP conformations as accessibility of the S-IIP is restricted only to the GDP-bound state. This strategy proved feasible for inhibiting mutant KRAS in vitro; however, it is uncertain whether this approach would translate to in vivo. Here, we describe structure-based design and identification of ARS-1620, a covalent compound with high potency and selectivity for KRASG12C. ARS-1620 achieves rapid and sustained in vivo target occupancy to induce tumor regression. We use ARS-1620 to dissect oncogenic KRAS dependency and demonstrate that monolayer culture formats significantly underestimate KRAS dependency in vivo. This study provides in vivo evidence that mutant KRAS can be selectively targeted and reveals ARS-1620 as representing a new generation of KRASG12C-specific inhibitors with promising therapeutic potential.

Helenalin Analogues Targeting NF-κB p65: Thiol Reactivity and Cellular Potency Studies of Varied Electrophiles Authors

Dr. John C. Widen, Dr. Aaron M. Kempema, Jordan W. Baur, Hannah M. Skopec, Jacob T. Edwards, Tenley J. Brown, Dr. Dennis A. Brown, Dr. Frederick A. Meece, Prof. Daniel A. Harki

Helenalin is a pseudoguaianolide natural product that targets Cys38 within the DNA binding domain of NF-κB transcription factor p65 (RelA). Helenalin contains two Michael acceptors that covalently modify cysteines: a α-methylene-γ-butyrolactone and a cyclopentenone. We recently reported two simplified helenalin analogues that mimic the biological activity of helenalin and contain both electrophilic moieties. To determine the individual contributions of the Michael acceptors toward NF-κB inhibition, we synthesized a small library of helenalin-based analogues containing various combinations of α-methylene-γ-butyrolactones and cyclopentenones. The kinetics of thiol addition to a subset of the analogues was measured to determine the relative thiol reactivities of the embedded electrophiles. Additionally, the cellular NF-κB inhibitory activities of the analogues were determined to elucidate the contributions of each Michael acceptor to biological potency. Our studies suggest the α-methylene-γ-butyrolactone contributes most significantly to the NF-κB inhibition of our simplified helenalin analogues.

Novel K-Ras G12C Switch-II covalent binders destabilize Ras and accelerate nucleotide exchange

Chimno Ihuoma Nnadi, Meredith L. Jenkins, Daniel R. Gentile, Leslie A. Bateman, Daniel Zaidman, Trent E. Balius, Daniel K. Nomura, John E. Burke, Kevan M. Shokat, and Nir London

J. Chem. Inf. Model., Just Accepted Manuscript
DOI: 10.1021/acs.jcim.7b00399

The success of targeted covalent inhibitors in the global pharmaceutical industry has led to a resurgence of covalent drug discovery. However, covalent inhibitor design for flexible binding sites remains a difficult task due to lack of methodological development. Here, we compared covalent docking to empirical electrophile screening, against the highly dynamic target K-RasG12C. While the overall hit-rate of both methods was comparable, we were able to rapidly progress a docking hit to a potent irreversible covalent inhibitor that modifies the inactive, GDP-bound state of K-RasG12C. Hydrogen-deuterium exchange mass spectrometry was used to probe the protein dynamics of compound binding to the switch-II pocket and subsequent destabilization of the nucleotide-binding region. SOS-mediated nucleotide exchange assays showed that, contrary to prior switch-II pocket inhibitors, these compounds appear to accelerate nucleotide exchange. This study highlights the efficiency of covalent docking as a tool for the discovery of chemically novel hits against challenging targets.

Progress with covalent small-molecule kinase inhibitors

Zheng Zhao, Philip E. Bourne

Drug Discovery Today, 2018

doi: 10.1016/j.drudis.2018.01.035

With reduced risk of toxicity and high selectivity, covalent small-molecule kinase inhibitors (CSKIs) have emerged rapidly. Through the lens of structural system pharmacology, here we review this rapid progress by considering design strategies and the challenges and opportunities offered by current CSKIs.