Chemoproteomic discovery of a covalent allosteric inhibitor of WRN helicase

Kristen A. Baltgalvis, Kelsey N. Lamb, Kent T. Symons, Chu-Chiao Wu, Melissa A. Hoffman, Aaron N. Snead, Xiaodan Song, Thomas Glaza, Shota Kikuchi, Jason C. Green, Donald C. Rogness, Betty Lam, Maria E. Rodriguez-Aguirre, David R. Woody, Christie L. Eissler, Socorro Rodiles, Seth M. Negron, Steffen M. Bernard, Eileen Tran, Jonathan Pollock, Ali Tabatabaei, Victor Contreras, Heather N. Williams, Martha K. Pastuszka, John J. Sigler, Piergiorgio Pettazzoni, Markus G. Rudolph, Moritz Classen, Doris Brugger, Christopher Claiborne, Jean-Marc Plancher, Isabel Cuartas, Joan Seoane, Laurence E. Burgess, Robert T. Abraham, David S. Weinstein, Gabriel M. Simon, Matthew P. Patricelli & Todd M. Kinsella

Nature, 2024

https://doi.org/10.1038/s41586-024-07318-y

WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1,2,3,4,5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.

DrugMap: A quantitative pan-cancer analysis of cysteine ligandability

Mariko Takahashi, Harrison B. Chong,Siwen Zhang, Tzu-Yi Yang,Matthew J. Lazarov,Stefan Harry,Michelle Maynard, Brendan Hilbert,Ryan D. White,Heather E. Murrey, Chih-Chiang Tsou, Kira Vordermark, Jonathan Assaad, Magdy Gohar, Benedikt R. Dürr et al.

Cell, 2024
DOI: https://doi.org/10.1016/j.cell.2024.03.027

Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors for a wide range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed “DrugMap,” an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NF-κB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NF-κB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription-factor activity.

Thiophene-fused γ-lactams inhibit the SARS-CoV-2 main protease via reversible covalent acylation

Gayatri Gayatri , Lennart Brewitz , Lewis Ibbotson , Eidarus Saleh , Shyam Basak , Hani Choudhry and Christopher J Schofield

Chem. Sci., 2024

DOI: 10.1039/D4SC01027B (Edge Article) 

Enzyme inhibitors working by O-acylation of nucleophilic serine residues are of immense medicinal importance, as exemplified by the β-lactam antibiotics. By contrast, inhibition of nucleophilic cysteine enzymes by S-acylation has not been widely exploited for medicinal applications. The SARS-CoV-2 main protease (Mpro) is a nucleophilic cysteine protease and a validated therapeutic target for COVID-19 treatment using small-molecule inhibitors. The clinically used Mpro inhibitors nirmatrelvir and simnotrelvir work via reversible covalent reaction of their electrophilic nitrile with the Mpro nucleophilic cysteine (Cys145). We report combined structure activity relationship and mass spectrometric studies revealing that appropriately functionalized γ-lactams can potently inhibit Mpro by reversible covalent reaction with Cys145 of Mpro. The results suggest that γ-lactams have potential as electrophilic warheads for development of covalently reacting small-molecule inhibitors of Mpro and, by implication, other nucleophilic cysteine enzymes.

Targeting KRAS Diversity: Covalent Modulation of G12X and Beyond in Cancer Therapy

Tonia Kirschner, Matthias P. Müller, and Daniel Rauh

Journal of Medicinal Chemistry 2024

DOI: 10.1021/acs.jmedchem.3c02403

The GTPase KRAS acts as a switch in cellular signaling, transitioning between inactive GDP-bound and active GTP-bound states. In about 20% of human cancers, oncogenic RAS mutations disrupt this balance, favoring the active form and promoting proliferative signaling, thus rendering KRAS an appealing target for precision medicine in oncology. In 2013, Shokat and co-workers achieved a groundbreaking feat by covalently targeting a previously undiscovered allosteric pocket (switch II pocket (SWIIP)) of KRASG12C. This breakthrough led to the development and approval of sotorasib (AMG510) and adagrasib (MRTX849), revolutionizing the treatment of KRASG12C-dependent lung cancer. Recent achievements in targeting various KRASG12X mutants, using SWIIP as a key binding pocket, are discussed. Insights from successful KRASG12C targeting informed the design of molecules addressing other mutations, often in a covalent manner. These findings offer promise for innovative approaches in addressing commonly occurring KRAS mutations such as G12D, G12V, G12A, G12S, and G12R in various cancers.

Vinylpyridine as a Tunable Covalent Warhead Targeting C797 in EGFR

Nils Pemberton, Nina Compagne, Argyrides Argyrou, Emma Evertsson, Anders Gunnarsson, Jason G. Kettle, Jonathan P. Orme, and Richard A. Ward

ACS Medicinal Chemistry Letters 2024

DOI: 10.1021/acsmedchemlett.3c00425

To further facilitate the discovery of cysteine reactive covalent inhibitors, there is a need to develop new reactive groups beyond the traditional acrylamide-type warheads. Herein we describe the design and synthesis of covalent EGFR inhibitors that use vinylpyridine as the reactive group. Structure-based design identified the quinazoline-containing vinylpyridine 6 as a starting point. Further modifications focused on reducing reactivity resulted in substituted vinyl compound 12, which shows high EGFR potency and good kinase selectivity, as well as significantly reduced reactivity compared to the starting compound 6, confirming that vinylpyridines can be applied as an alternative cysteine reactive warhead with tunable reactivity.

Fitting of kinact and KI Values from Endpoint Pre-incubation IC50 Data

Lavleen K. Mader and Jeffrey W. Keillor

ACS Medicinal Chemistry Letters 2024

DOI: 10.1021/acsmedchemlett.4c00054

Experiments comprising a “pre-incubation” phase, where enzyme is incubated with inhibitor prior to the addition of assay substrate, are commonly used to evaluate covalent inhibitors, often via discontinuous or “endpoint” IC50 assays. However, due to the lack of mathematical tools to describe its biphasic time-dependent nature, this experiment has thus far been unable to provide kinact and KI values. Herein we report EPIC-Fit, a new method to determine kinact and KI values from global fitting of Endpoint Pre-incubation IC50 data that can be implemented using Microsoft Excel. Experimental characterization of a known tissue transglutaminase inhibitor, AA9, using EPIC-Fit provided kinact and KI values with strong correlations to the values determined by other, previously established methods of evaluation. This unprecedented method serves to finally include time-dependent pre-incubation endpoint assays in the medicinal chemist’s toolbox for rigorous characterization of irreversible inhibitors.

Proteomic Ligandability Maps of Spirocycle Acrylamide Stereoprobes Identify Covalent ERCC3 Degraders

Zhonglin Liu, Jarrett R. Remsberg, Haoxin Li, Evert Njomen, Kristen E. DeMeester, Yongfeng Tao, Guoqin Xia, Rachel E. Hayward, Minjin Yoo, Tracey Nguyen, Gabriel M. Simon, Stuart L. Schreiber, Bruno Melillo, and Benjamin F. Cravatt

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.3c13448

Covalent chemistry coupled with activity-based protein profiling (ABPP) offers a versatile way to discover ligands for proteins in native biological systems. Here, we describe a set of stereo- and regiochemically defined spirocycle acrylamides and the analysis of these electrophilic “stereoprobes” in human cancer cells by cysteine-directed ABPP. Despite showing attenuated reactivity compared to structurally related azetidine acrylamide stereoprobes, the spirocycle acrylamides preferentially liganded specific cysteines on diverse protein classes. One compound termed ZL-12A promoted the degradation of the TFIIH helicase ERCC3. Interestingly, ZL-12A reacts with the same cysteine (C342) in ERCC3 as the natural product triptolide, which did not lead to ERCC3 degradation but instead causes collateral loss of RNA polymerases. ZL-12A and triptolide cross-antagonized one another’s protein degradation profiles. Finally, we provide evidence that the antihypertension drug spironolactone─previously found to promote ERCC3 degradation through an enigmatic mechanism─also reacts with ERCC3_C342. Our findings thus describe monofunctional degraders of ERCC3 and highlight how covalent ligands targeting the same cysteine can produce strikingly different functional outcomes.

Reversible Covalent Inhibition─Desired Covalent Adduct Formation by Mass Action

Disha Patel, Zil E Huma, and Dustin Duncan

ACS Chemical Biology 2024

DOI: 10.1021/acschembio.3c00805

Covalent inhibition has seen a resurgence in the last several years. Although long-plagued by concerns of off-target effects due to nonspecific reactions leading to covalent adducts, there has been success in developing covalent inhibitors, especially within the field of anticancer therapy. Covalent inhibitors can have an advantage over noncovalent inhibitors since the formation of a covalent adduct may serve as an additional mode of selectivity due to the intrinsic reactivity of the target protein that is absent in many other proteins. Unfortunately, many covalent inhibitors form irreversible adducts with off-target proteins, which can lead to considerable side-effects. By designing the inhibitor to form reversible covalent adducts, one can leverage competing on/off kinetics in complex formation by taking advantage of the law of mass action. Although covalent adducts do form with off-target proteins, the reversible nature of inhibition prevents accumulation of the off-target adduct, thus limiting side-effects. In this perspective, we outline important characteristics of reversible covalent inhibitors, including examples and a guide for inhibitor development.

Azapeptides with unique covalent warheads as SARS-CoV-2 main protease inhibitors

Kaustav Khatua, Yugendar R. Alugubelli , Kai S. Yang, Veerabhadra R. Vulupala, Lauren R. Blankenship , Demonta Coleman, Sandeep Atla , Sankar P. Chaki, Zhi Zachary Geng , Xinyu R. Ma , Jing Xiao , Peng-Hsun Chen , Chia-Chuan D. Cho, Shivangi Sharma, Erol C. Vatansever, Yuying Ma, Ge Yu, Benjamin W. Neuman, Shiqing Xu , Wenshe Ray Liu

Antiviral Research, 225, 2024, 105874

https://doi.org/10.1016/j.antiviral.2024.105874

The main protease (MPro) of SARS-CoV-2, the causative agent of COVID-19, is a pivotal nonstructural protein critical for viral replication and pathogenesis. Its protease function relies on three active site pockets for substrate recognition and a catalytic cysteine for enzymatic activity. To develop potential SARS-CoV-2 antivirals, we successfully synthesized a diverse range of azapeptide inhibitors with various covalent warheads to target MPro's catalytic cysteine. Our characterization identified potent MPro inhibitors, including MPI89 that features an aza-2,2-dichloroacetyl warhead with a remarkable EC50 value of 10 nM against SARS-CoV-2 infection in ACE2+ A549 cells and a selective index of 875. MPI89 is also remarkably selective and shows no potency against SARS-CoV-2 papain-like protease and several human proteases. Crystallography analyses demonstrated that these inhibitors covalently engaged the catalytic cysteine and used the aza-amide carbonyl oxygen to bind to the oxyanion hole. MPI89 stands as one of the most potent MPro inhibitors, suggesting the potential for further exploration of azapeptides and the aza-2,2-dichloroacetyl warhead for developing effective therapeutics against COVID-19.

Discovery of α-Amidobenzylboronates as Highly Potent Covalent Inhibitors of Plasma Kallikrein

Matthew Allison, Rebecca L. Davie, Adrian J. Mogg, Sally L. Hampton, Jonas Emsley, and Michael J. Stocks

ACS Medicinal Chemistry Letters 2024

DOI: 10.1021/acsmedchemlett.3c00572

Hereditary angioedema (HAE), a rare genetic disorder, is associated with uncontrolled plasma kallikrein (PKa) enzyme activity leading to the generation of bradykinin swelling in subcutaneous and submucosal membranes in various locations of the body. Herein, we describe a series of potent α-amidobenzylboronates as potential covalent inhibitors of PKa. These compounds exhibited time-dependent inhibition of PKa (compound 20 IC50 66 nM at 1 min, 70 pM at 24 h). Further compound dissociation studies demonstrated that 20 showed no apparent reversibility comparable to d-Phe-Pro-Arg-chloromethylketone (PPACK) (23), a known nonselective covalent PKa inhibitor.

CHARMM-GUI PDB Reader and Manipulator: Covalent Ligand Modeling and Simulation

 Lingyang Kong, Sang-Jun Park Wonpil Im

Journal of Molecular Biology, 2024

https://doi.org/10.1016/j.jmb.2024.168554

Molecular modeling and simulation serve an important role in exploring biological functions of proteins at the molecular level, which is complementary to experiments. CHARMM-GUI (https://www.charmm-gui.org) is a web-based graphical user interface that generates molecular simulation systems and input files, and we have been continuously developing and extending its functionalities to facilitate various complex modeling and make molecular dynamics simulations more accessible to the scientific community. Currently, covalent drug discovery emerges as a popular and important field. Covalent drug forms a chemical bond with specific residues on the target protein, and it has advantages in potency for its prolonged inhibition effects. Even though there are higher demands in modeling PDB protein structures with various covalent ligand types, proper modeling of covalent ligands remains challenging. This work presents a new functionality in CHARMM-GUI PDB Reader & Manipulator that can handle a diversity of ligand-amino acid linkage types, which is validated by a careful benchmark study using over 1,000 covalent ligand structures in RCSB PDB. We hope that this new functionality can boost the modeling and simulation study of covalent ligands.

Restricted Rotational Flexibility of the C5α-Methyl-Substituted Carbapenem NA-1-157 Leads to Potent Inhibition of the GES-5 Carbapenemase

Nichole K. Stewart, Marta Toth, Pojun Quan, Michael Beer, John D. Buynak, Clyde A. Smith, and Sergei B. Vakulenko

ACS Infectious Diseases 2024

DOI: 10.1021/acsinfecdis.3c00683

Carbapenem antibiotics are used as a last-resort treatment for infections caused by multidrug-resistant bacteria. The wide spread of carbapenemases in Gram-negative bacteria has severely compromised the utility of these drugs and represents a serious public health threat. To combat carbapenemase-mediated resistance, new antimicrobials and inhibitors of these enzymes are urgently needed. Here, we describe the interaction of the atypically C5α-methyl-substituted carbapenem, NA-1-157, with the GES-5 carbapenemase. MICs of this compound against Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii producing the enzyme were reduced 4–16-fold when compared to MICs of the commercial carbapenems, reaching clinically sensitive breakpoints. When NA-1-157 was combined with meropenem, a strong synergistic effect was observed. Kinetic and ESI-LC/MS studies demonstrated that NA-1-157 is a potent inhibitor of GES-5, with a high inactivation efficiency of (2.9 ± 0.9) × 105 M–1 s–1. Acylation of GES-5 by NA-1-157 was biphasic, with the fast phase completing within seconds, and the slow phase taking several hours and likely proceeding through a reversible tetrahedral intermediate. Deacylation was extremely slow (k3 = (2.4 ± 0.3) × 10–7 s–1), resulting in a residence time of 48 ± 6 days. MD simulation of the GES-5-meropenem and GES-5-NA-1-157 acyl-enzyme complexes revealed that the C5α-methyl group in NA-1-157 sterically restricts rotation of the 6α-hydroxyethyl group preventing ingress of the deacylating water into the vicinity of the scissile bond of the acyl-enzyme intermediate. These data demonstrate that NA-1-157 is a potent irreversible inhibitor of the GES-5 carbapenemase.

Discovery of a Covalent Inhibitor Selectively Targeting the Autophosphorylation Site of c-Src Kinase

Huimin Zhang, Dounan Xu, Hongchan Huang, Hao Jiang, Linghao Hu, Liping Liu, Ge Sun, Jing Gao, Yuanqing Li, Cuicui Xia, Shijie Chen, Hu Zhou, Xiangqian Kong, Mingliang Wang, and Cheng Luo

ACS Chemical Biology 2024

DOI: 10.1021/acschembio.4c00048

Nonreceptor tyrosine kinase c-Src plays a crucial role in cell signaling and contributes to tumor progression. However, the development of selective c-Src inhibitors turns out to be challenging. In our previous study, we performed posttranslational modification-inspired drug design (PTMI-DD) to provide a plausible way for designing selective kinase inhibitors. In this study, after identifying a unique pocket comprising a less conserved cysteine and an autophosphorylation site in c-Src as well as a promiscuous covalent inhibitor, chemical optimization was performed to obtain (R)-LW-Srci-8 with nearly 75-fold improved potency (IC50 = 35.83 ± 7.21 nM). Crystallographic studies revealed the critical C–F···C═O interactions that may contribute to tight binding. The kinact and Ki values validated the improved binding affinity and decreased warhead reactivity of (R)-LW-Srci-8 for c-Src. Notably, in vitro tyrosine kinase profiling and cellular activity-based protein profiling (ABPP) cooperatively indicated a specific inhibition of c-Src by (R)-LW-Srci-8. Intriguingly, (R)-LW-Srci-8 preferentially binds to inactive c-Src with unphosphorylated Y419 both in vitro and in cells, subsequently disrupting the autophosphorylation. Collectively, our study demonstrated the feasibility of developing selective kinase inhibitors by cotargeting a nucleophilic residue and a posttranslational modification site and providing a chemical probe for c-Src functional studies.

Redirecting the pioneering function of FOXA1 with covalent small molecules

Sang Joon Won, Yuxiang Zhang, Christopher J. Reinhardt, Nicole S. MacRae, Kristen E. DeMeester, Evert Njomen, Lauren M. Hargis, Jarrett R. Remsberg, Bruno Melillo, Benjamin F. Cravatt, Michael A. Erb

bioRxiv 2024.03.21.586158; doi: https://doi.org/10.1101/2024.03.21.586158

Pioneer transcription factors (TFs) exhibit a specialized ability to bind to and open closed chromatin, facilitating engagement by other regulatory factors involved in gene activation or repression. Chemical probes are lacking for pioneer TFs, which has hindered their mechanistic investigation in cells. Here, we report the chemical proteomic discovery of electrophilic small molecules that stereoselectively and site-specifically bind the pioneer TF, FOXA1, at a cysteine (C258) within the forkhead DNA-binding domain. We show that these covalent ligands react with FOXA1 in a DNA-dependent manner and rapidly remodel its pioneer activity in prostate cancer cells reflected in redistribution of FOXA1 binding across the genome and directionally correlated changes in chromatin accessibility. Motif analysis supports a mechanism where the covalent ligands relax the canonical DNA binding preference of FOXA1 by strengthening interactions with suboptimal ancillary sequences in predicted proximity to C258. Our findings reveal a striking plasticity underpinning the pioneering function of FOXA1 that can be controlled by small molecules.

Advancing protein therapeutics through proximity-induced chemistry

Linqi Cheng Yixian Wang, Yiming Guo, Sophie S. Zhang Han Xiao
Cell Chemical Biology, 2024 Volume 31,  3, 428 - 445

https://doi.org/10.1016/j.chembiol.2023.09.004

Recent years have seen a remarkable growth in the field of protein-based medical treatments. Nevertheless, concerns have arisen regarding the cytotoxicity limitations, low affinity, potential immunogenicity, low stability, and challenges to modify these proteins. To overcome these obstacles, proximity-induced chemistry has emerged as a next-generation strategy for advancing protein therapeutics. This method allows site-specific modification of proteins with therapeutic agents, improving their effectiveness without extensive engineering. In addition, this innovative approach enables spatial control of the reaction based on proximity, facilitating the formation of irreversible covalent bonds between therapeutic proteins and their targets. This capability becomes particularly valuable in addressing challenges such as the low affinity frequently encountered between therapeutic proteins and their targets, as well as the limited availability of small molecules for specific protein targets. As a result, proximity-induced chemistry is reshaping the field of protein drug preparation and propelling the revolution in novel protein therapeutics.

Molecular Insights into the Impact of Mutations on the Binding Affinity of Targeted Covalent Inhibitors of BTK

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

The Journal of Physical Chemistry B 2024

DOI: 10.1021/acs.jpcb.4c00310

Targeted covalent inhibitors (TCIs) have witnessed a significant resurgence in recent years, particularly in the kinase drug discovery field for treating diverse clinical indications. The inhibition of Bruton’s tyrosine kinase (BTK) for treating B-cell cancers is a classic example where TCIs such as ibrutinib have had breakthroughs in targeted therapy. However, selectivity remains challenging, and the emergence of resistance mutations is a critical concern for clinical efficacy. Computational methods that can accurately predict the impact of mutations on inhibitor binding affinity could prove helpful in informing targeted approaches─providing insights into drug resistance mechanisms. In addition, such systems could help guide the systematic evaluation and impact of mutations in disease models for optimal experimental design. Here, we have employed in silico physics-based methods to understand the effects of mutations on the binding affinity and conformational dynamics of select TCIs of BTK. The TCIs studied include ibrutinib, acalabrutinib, and zanubrutinib─all of which are FDA-approved drugs for treating multiple forms of leukemia and lymphoma. Our results offer useful molecular insights into the structural determinants, thermodynamics, and conformational energies that impact ligand binding for this biological target of clinical relevance.

Covalent Fragment Screening and Optimization Identifies the Chloroacetohydrazide Scaffold as Inhibitors for Ubiquitin C-terminal Hydrolase L1

Ryan D. Imhoff, Rishi Patel, Muhammad Hassan Safdar, Hannah B. L. Jones, Adan Pinto-Fernandez, Iolanda Vendrell, Hao Chen, Christine S. Muli, Aaron D. Krabill, Benedikt M. Kessler, Michael K. Wendt, Chittaranjan Das, and Daniel P. Flaherty

Journal of Medicinal Chemistry 2024

DOI: 10.1021/acs.jmedchem.3c01661

Dysregulation of the ubiquitin-proteasome systems is a hallmark of various disease states including neurodegenerative diseases and cancer. Ubiquitin C-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme, is expressed primarily in the central nervous system under normal physiological conditions, however, is considered an oncogene in various cancers, including melanoma, lung, breast, and lymphoma. Thus, UCHL1 inhibitors could serve as a viable treatment strategy against these aggressive cancers. Herein, we describe a covalent fragment screen that identified the chloroacetohydrazide scaffold as a covalent UCHL1 inhibitor. Subsequent optimization provided an improved fragment with single-digit micromolar potency against UCHL1 and selectivity over the closely related UCHL3. The molecule demonstrated efficacy in cellular assays of metastasis. Additionally, we report a ligand-bound crystal structure of the most potent molecule in complex with UCHL1, providing insight into the binding mode and information for future optimization.

An orally bioavailable SARS-CoV-2 main protease inhibitor exhibits improved affinity and reduced sensitivity to mutations

Michael Westberg et al.

Sci. Transl. Med.16,eadi0979(2024).

DOI:10.1126/scitranslmed.adi0979

Inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) such as nirmatrelvir (NTV) and ensitrelvir (ETV) have proven effective in reducing the severity of COVID-19, but the presence of resistance-conferring mutations in sequenced viral genomes raises concerns about future drug resistance. Second-generation oral drugs that retain function against these mutants are thus urgently needed. We hypothesized that the covalent hepatitis C virus protease inhibitor boceprevir (BPV) could serve as the basis for orally bioavailable drugs that inhibit SARS-CoV-2 Mpro more efficiently than existing drugs. Performing structure-guided modifications of BPV, we developed a picomolar-affinity inhibitor, ML2006a4, with antiviral activity, oral pharmacokinetics, and therapeutic efficacy similar or superior to those of NTV. A crucial feature of ML2006a4 is a derivatization of the ketoamide reactive group that improves cell permeability and oral bioavailability. Last, ML2006a4 was found to be less sensitive to several mutations that cause resistance to NTV or ETV and occur in the natural SARS-CoV-2 population. Thus, anticipatory design can preemptively address potential resistance mechanisms to expand future treatment options against coronavirus variants.

Strain-release alkylation of Asp12 enables mutant selective targeting of K-Ras-G12D

Qinheng Zheng, Ziyang Zhang, Keelan Z. Guiley & Kevan M. Shokat

Nat Chem Biol 2024

 https://doi.org/10.1038/s41589-024-01565-w

K-Ras is the most commonly mutated oncogene in human cancer. The recently approved non-small cell lung cancer drugs sotorasib and adagrasib covalently capture an acquired cysteine in K-Ras-G12C mutation and lock it in a signaling-incompetent state. However, covalent inhibition of G12D, the most frequent K-Ras mutation particularly prevalent in pancreatic ductal adenocarcinoma, has remained elusive due to the lack of aspartate-targeting chemistry. Here we present a set of malolactone-based electrophiles that exploit ring strain to crosslink K-Ras-G12D at the mutant aspartate to form stable covalent complexes. Structural insights from X-ray crystallography and exploitation of the stereoelectronic requirements for attack of the electrophile allowed development of a substituted malolactone that resisted attack by aqueous buffer but rapidly crosslinked with the aspartate-12 of K-Ras in both GDP and GTP state. The GTP-state targeting allowed effective suppression of downstream signaling, and selective inhibition of K-Ras-G12D-driven cancer cell proliferation in vitro and xenograft growth in mice.

Chemical tools to expand the ligandable proteome: diversity-oriented synthesis-based photoreactive stereoprobes

Daisuke Ogasawara, David Konrad, Zher Yin Tan, Kimberly Carey, Jessica Luo, Sang Joon Won, Haoxin Li, Trever Carter, Kristen DeMeester, Evert Njomen, Stuart Schreiber, Ramnik Xavier, Bruno Melillo, Benjamin Cravatt

bioRxiv 2024.02.27.582206; 

doi: https://doi.org/10.1101/2024.02.27.582206

Chemical proteomics enables the global assessment of small molecule-protein interactions in native biological systems and has emerged as a versatile approach for ligand discovery. The range of small molecules explored by chemical proteomics has, however, been limited. Here, we describe a diversity-oriented synthesis (DOS)-inspired library of stereochemically-defined compounds bearing diazirine and alkyne units for UV light-induced covalent modification and click chemistry enrichment of interacting proteins, respectively. We find that these photo-stereoprobes interact in a stereoselective manner with hundreds of proteins from various structural and functional classes in human cells and demonstrate that these interactions can form the basis for high-throughput screening-compatible nanoBRET assays. Integrated phenotypic analysis and chemical proteomics identified photo-stereoprobes that modulate autophagy by engaging the mitochondrial serine protease CLPP. Our findings show the utility of photo-stereoprobes for expanding the ligandable proteome, furnishing target engagement assays, and discovering and characterizing bioactive small molecules by cell-based screening.

Formaldehyde regulates S-adenosylmethionine biosynthesis and one-carbon metabolism

VANHA N. PHAM, KEVIN J. BRUEMMER, JOEL D. W. TOH, EVA J. GE, LOGAN TENNEY, CARL C. WARD, FELIX A. DINGLER, CHRISTOPHER L. MILLINGTON, CARLOS A. GARCIA-PRIETO  MIA C. PULOS-HOLMES  NICHOLAS T. INGOLIA LUCAS B. PONTEL MANEL ESTELLER  KETAN J. PATEL  DANIEL K. NOMURA AND CHRISTOPHER J. CHANG 

Science 2023 382, 6670

DOI: 10.1126/science.abp9201

INTRODUCTION

One-carbon metabolism manages cellular carbon pools by detoxifying highly reactive carbon species, such as aldehydes, and diverting their carbon toward the biosynthesis of useful products, including amino acids and nucleotides. Formaldehyde (FA) is a major one-carbon unit derived from exogenous environmental exposure and endogenous sources and is quickly scavenged in the cell through enzymatic oxidation to formate and carbon dioxide and/or metabolized through the folate cycle. S-adenosylmethionine (SAM) serves as the primary cellular methyl donor and harbors one-carbon units in a stable and accessible form. The ability to decipher the biochemical interplay between toxic reactive carbon species and stable physiological carbon units is essential for understanding fundamentals of one-carbon metabolism across all kingdoms of life. Especially important is understanding how aberrant carbon imbalances are connected to human diseases such as cancer, liver diseases, and asthma. Although the chronic exposure of toxic aldehydes is correlated to disease states, biological mechanisms of aldehyde signaling and their relation to carbon metabolism remain underexplored.

RATIONALE

Owing to its highly electrophilic nature, we hypothesized that FA could act as a one-carbon signal sensed by privileged cysteine sites across the proteome. FA reacts with cysteines on synthetic peptides, and we designed an unbiased, proteome-wide profiling study to systematically identify FA-sensitive cysteine residues. This work builds a biochemical framework for understanding global FA reactivity as a selective posttranslational modification of target proteins and downstream regulatory effects of such modifications.

RESULTS

Activity-based protein profiling identified FA modification of privileged cysteine sites across the proteome, including several enzymes responsible for FA metabolism, one-carbon metabolism, and amino acid biosynthesis. We focused on biochemical characterization of a key Cys120 residue on the SAM-generating enzyme S-adenosylmethionine synthase isoform type-1 (MAT1A) that is proximal to the MAT1A active site. FA exposure resulted in inhibition of MAT1A activity in an isoform-specific manner, which led to decreased SAM production. Cellular models containing only the MAT1A isoform displayed a reciprocal decrease in SAM levels with increasing doses of FA exposure. Moreover, an Adh5–/– mouse model of chronic FA elevation also showed SAM deficiency accompanied by lower levels of methylation on select histone methyl sinks. The chronic FA model also resulted in a decrease in methylation of the Mat1a promoter region, resulting in increased MAT1A expression as a compensatory mechanism to maintain available carbon units. We deciphered a compensatory biochemical feedback cycle where FA-dependent SAM deficiency led to an increase in MAT1A expression through genetic and epigenetic mechanisms regulated by FA-dependent transcription factors and DNA promoter hypomethylation, respectively.

CONCLUSION

In contrast to the traditional view of FA as an indiscriminate electrophile and toxic metabolite, we show that FA is sensed by specific cysteine sites in the proteome to regulate one-carbon metabolism feedback cycles through SAM biosynthesis. FA reacts with a key cysteine residue on MAT1A to inhibit its activity, resulting in SAM depletion and downstream changes in histone and DNA methylation. Under normal homeostatic conditions, FA is quickly sequestered into the folate cycle for conservation of one-carbon units to maintain balanced SAM biosynthesis. In response to FA overload, reciprocal SAM depletion through isoform-specific MAT1A inhibition results in changes to cellular methylation potential, epigenetic dysregulation, and perturbations in one-carbon metabolism, which in turns leads to compensatory up-regulation of MAT1A expression. This work provides a starting point for further exploration of aldehydes as signaling agents and the nexus between one-carbon metabolism and one-carbon signaling.

Molecular Bidents with Two Electrophilic Warheads as a New Pharmacological Modality

Zhengnian Li, Jie Jiang, Scott B. Ficarro, Tyler S. Beyett, Ciric To, Isidoro Tavares, Yingde Zhu, Jiaqi Li, Michael J. Eck, Pasi A. Jänne, Jarrod A. Marto, Tinghu Zhang, Jianwei Che, and Nathanael S. Gray

ACS Central Science 2024

DOI: 10.1021/acscentsci.3c01245

A systematic strategy to develop dual-warhead inhibitors is introduced to circumvent the limitations of conventional covalent inhibitors such as vulnerability to mutations of the corresponding nucleophilic residue. Currently, all FDA-approved covalent small molecules feature one electrophile, leaving open a facile route to acquired resistance. We conducted a systematic analysis of human proteins in the protein data bank to reveal ∼400 unique targets amendable to dual covalent inhibitors, which we term “molecular bidents”. We demonstrated this strategy by targeting two kinases: MKK7 and EGFR. The designed compounds, ZNL-8162 and ZNL-0056, are ATP-competitive inhibitors that form two covalent bonds with cysteines and retain potency against single cysteine mutants. Therefore, molecular bidents represent a new pharmacological modality with the potential for improved selectivity, potency, and drug resistance profile.

Development of Oxadiazolone Activity-Based Probes Targeting FphE for Specific Detection of Staphylococcus aureus Infections

Jeyun Jo, Tulsi Upadhyay, Emily C. Woods, Ki Wan Park, Nichole J. Pedowitz, Joanna Jaworek-Korjakowska, Sijie Wang, Tulio A. Valdez, Matthias Fellner, and Matthew Bogyo

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.3c13974

Staphylococcus aureus (S. aureus) is a major human pathogen that is responsible for a wide range of systemic infections. Since its propensity to form biofilms in vivo poses formidable challenges for both detection and treatment, tools that can be used to specifically image S. aureus biofilms are highly valuable for clinical management. Here, we describe the development of oxadiazolone-based activity-based probes to target the S. aureus-specific serine hydrolase FphE. Because this enzyme lacks homologues in other bacteria, it is an ideal target for selective imaging of S. aureus infections. Using X-ray crystallography, direct cell labeling, and mouse models of infection, we demonstrate that oxadiazolone-based probes enable specific labeling of S. aureus bacteria through the direct covalent modification of the FphE active site serine. These results demonstrate the utility of the oxadizolone electrophile for activity-based probes and validate FphE as a target for the development of imaging contrast agents for the rapid detection of S. aureus infections.

A covalent compound selectively inhibits RNA demethylase ALKBH5 rather than FTO

Gan-Qiang Lai,  Yali Li,  Heping Zhu,   Tao Zhang,  Jing Gao,  Hu Zhou  and  Cai-Guang Yang 

RSC Chem. Biol., 2024, DOI: 10.1039/D3CB00230F

N6-Methyladenosine (m6A) is the most prevalent mRNA modification and is required for gene regulation in eukaryotes. ALKBH5, an m6A demethylase, is a promising target, particularly for anticancer drug discovery. However, the development of selective and potent inhibitors of ALKBH5 rather than FTO remains challenging. Herein, we used a targeted covalent inhibition strategy and identified a covalent inhibitor, TD19, which selectively inhibits ALKBH5 compared with FTO demethylase in protein-based and tumor cell-based assays. TD19 irreversibly modifies the residues C100 and C267, preventing ALKBH5 from binding to m6A-containing RNA. Moreover, TD19 displays good anticancer efficacy in acute myeloid leukemia and glioblastoma multiforme cell lines. Thus, the ALKBH5 inhibitor developed in this study, which selectively targets ALKBH5 compared with FTO, can potentially be used as a probe for investigating the biological functions of RNA demethylase and as a lead compound in anticancer research.

Discovery of 6-Formylpyridyl Urea Derivatives as Potent Reversible-Covalent Fibroblast Growth Factor Receptor 4 Inhibitors with Improved Anti-Hepatocellular Carcinoma Activity

Fang Yang, Qianmeng Lin, Xiaojuan Song, Huisi Huang, Xiaojuan Chen, Jianwen Tan, Yun Li, Yang Zhou, Zhengchao Tu, Hongli Du, Zhi-min Zhang, Raquel Ortega, Xiaojing Lin, Adam V. Patterson, Jeff B. Smaill, Yongheng Chen, and Xiaoyun Lu

Journal of Medicinal Chemistry 2024

DOI: 10.1021/acs.jmedchem.3c01810

Fibroblast growth factor receptor 4 (FGFR4) has been considered as a potential anticancer target due to FGF19/FGFR4 mediated aberrant signaling in hepatocellular carcinoma (HCC). Several FGFR4 inhibitors have been reported, but none have gained approval. Herein, a series of 5-formyl-pyrrolo[3,2-b]pyridine-3-carboxamides and a series of 6-formylpyridyl ureas were characterized as selective reversible-covalent FGFR4 inhibitors. The representative 6-formylpyridyl urea 8z exhibited excellent potency against FGFR4WT, FGFR4V550L, and FGFR4V550M with IC50 values of 16.3, 12.6, and 57.3 nM, respectively. It also potently suppressed proliferation of Ba/F3 cells driven by FGFR4WT, FGFR4V550L, and FGFR4V550M, and FGFR4-dependent Hep3B and Huh7 HCC cells, with IC50 values of 1.2, 13.5, 64.5, 15.0, and 20.4 nM, respectively. Furthermore, 8z displayed desirable microsomal stability and significant in vivo efficacy in the Huh7 HCC cancer xenograft model in nude mice. The study provides a promising new lead for anticancer drug discovery directed toward overcoming FGFR4 gatekeeper mutation mediated resistance in HCC patients.

Structure-based design of a phosphotyrosine-masked covalent ligand targeting the E3 ligase SOCS2

Sarath Ramachandran, Nikolai Makukhin, Kevin Haubrich, Manjula Nagala, Beth Forrester, Dylan M. Lynch, Ryan Casement, Andrea Testa, Elvira Bruno, Rosaria Gitto & Alessio Ciulli 

Nat Commun 14, 6345 (2023). 

https://doi.org/10.1038/s41467-023-41894-3

The Src homology 2 (SH2) domain recognizes phosphotyrosine (pY) post translational modifications in partner proteins to trigger downstream signaling. Drug discovery efforts targeting the SH2 domains have long been stymied by the poor drug-like properties of phosphate and its mimetics. Here, we use structure-based design to target the SH2 domain of the E3 ligase suppressor of cytokine signaling 2 (SOCS2). Starting from the highly ligand-efficient pY amino acid, a fragment growing approach reveals covalent modification of Cys111 in a co-crystal structure, which we leverage to rationally design a cysteine-directed electrophilic covalent inhibitor MN551. We report the prodrug MN714 containing a pivaloyloxymethyl (POM) protecting group and evidence its cell permeability and capping group unmasking using cellular target engagement and in-cell 19F NMR spectroscopy. Covalent engagement at Cys111 competitively blocks recruitment of cellular SOCS2 protein to its native substrate. The qualified inhibitors of SOCS2 could find attractive applications as chemical probes to understand the biology of SOCS2 and its CRL5 complex, and as E3 ligase handles in proteolysis targeting chimera (PROTACs) to induce targeted protein degradation.

Development of a Covalent Small Molecule Downmodulator for the Transcription Factor Brachyury

Davis H. Chase, Adrian M. Bebenek, Pengju Nie, Saul Jaime-Figueroa, Arseniy Butrin, Danielle A. Castro, John Hines, Brian M. Linhares, Craig M. Crews

Angew. Chem. Int. Ed. 2024, e202316496.

https://doi.org/10.1002/anie.202316496

Brachyury is an oncogenic transcription factor whose overexpression drives chordoma growth. The downmodulation of brachyury in chordoma cells has demonstrated therapeutic potential, however, as a transcription factor it is classically deemed “undruggable”.  Given that direct pharmacological intervention against brachyury has proven difficult, attempts at intervention have instead targeted upstream kinases. Recently, afatinib, an FDA-approved kinase inhibitor, has been shown to modulate brachyury levels in multiple chordoma cell lines. Herein, we use afatinib as a lead to undertake a structure-based drug design approach, aided by mass-spectrometry and x-ray crystallography, to develop DHC-156, a small molecule that more selectively binds brachyury and downmodulates it as potently as afatinib. We eliminated kinase-inhibition from this novel scaffold while demonstrating that DHC-156 induces the post-translational downmodulation of brachyury that results in an irreversible impairment of chordoma tumor cell growth. In doing so, we demonstrate the feasibility of direct brachyury modulation, which may further be developed into more potent tool compounds and therapies.

Mixed Alkyl/Aryl Phosphonates Identify Metabolic Serine Hydrolases as Antimalarial Targets

John M. Bennett, Sunil K. Narwal, Stephanie Kabeche, Daniel Abegg, Fiona Hackett, Tomas Yeo, Veronica L. Li, Ryan K. Muir, Franco F. Faucher, Scott Lovell, Michael J. Blackman, Alexander Adibekian, Ellen Yeh, David A. Fidock, Matthew Bogyo

bioRxiv 2024.01.11.575224; doi: https://doi.org/10.1101/2024.01.11.575224

Malaria, caused by Plasmodium falciparum, remains a significant health burden. A barrier for developing anti-malarial drugs is the ability of the parasite to rapidly generate resistance. We demonstrated that Salinipostin A (SalA), a natural product, kills parasites by inhibiting multiple lipid metabolizing serine hydrolases, a mechanism with a low propensity for resistance. Given the difficulty of employing natural products as therapeutic agents, we synthesized a library of lipidic mixed alkyl/aryl phosphonates as bioisosteres of SalA. Two constitutional isomers exhibited divergent anti-parasitic potencies which enabled identification of therapeutically relevant targets. We also confirm that this compound kills parasites through a mechanism that is distinct from both SalA and the pan-lipase inhibitor, Orlistat. Like SalA, our compound induces only weak resistance, attributable to mutations in a single protein involved in multidrug resistance. These data suggest that mixed alkyl/aryl phosphonates are a promising, synthetically tractable anti-malarials with a low-propensity to induce resistance.

Landscape of In Silico Tools for Modeling Covalent Modification of Proteins: A Review on Computational Covalent Drug Discovery

Md Nazmul Hasan, Manisha Ray, and Arjun Saha

The Journal of Physical Chemistry B 2023 127 (45), 9663-9684

DOI: 10.1021/acs.jpcb.3c04710

Covalent drug discovery has been a challenging research area given the struggle of finding a sweet balance between selectivity and reactivity for these drugs, the lack of which often leads to off-target activities and hence undesirable side effects. However, there has been a resurgence in covalent drug design following the success of several covalent drugs such as boceprevir (2011), ibrutinib (2013), neratinib (2017), dacomitinib (2018), zanubrutinib (2019), and many others. Design of covalent drugs includes many crucial factors, where “evaluation of the binding affinity” and “a detailed mechanistic understanding on covalent inhibition” are at the top of the list. Well-defined experimental techniques are available to elucidate these factors; however, often they are expensive and/or time-consuming and hence not suitable for high throughput screens. Recent developments in in silico methods provide promise in this direction. In this report, we review a set of recent publications that focused on developing and/or implementing novel in silico techniques in “Computational Covalent Drug Discovery (CCDD)”. We also discuss the advantages and disadvantages of these approaches along with what improvements are required to make it a great tool in medicinal chemistry in the near future.

Discovery of lirafugratinib (RLY-4008), a highly selective irreversible small-molecule inhibitor of FGFR2

Schönherr, H.; Ayaz, P.; Taylor, A. M.; Casaletto, J. B.; Touré, B. B.; Moustakas, D. T.; Hudson, B. M.; Valverde, R.; Zhao, S.; O’Hearn, P. J.; Foster, L.; Sharon, D. A.; Garfinkle, S.; Giordanetto, F.; Lescarbeau, A.; Kurukulasuriya, R.; Gerami-Moayed, N.; Maglic, D.; Bruderek, K.; Naik, G.; Gunaydin, H.; Mader, M. M.; Boezio, A. A.; McLean, T. H.; Chen, R.; Wang, Y.; Shaw, D. E.; Watters, J.; Bergstrom, D. A.

Proceedings of the National Academy of Sciences 2024, 121 (6), e2317756121. https://doi.org/10.1073/pnas.2317756121.

Fibroblast growth factor receptor (FGFR) kinase inhibitors have been shown to be effective in the treatment of intrahepatic cholangiocarcinoma and other advanced solid tumors harboring FGFR2 alterations, but the toxicity of these drugs frequently leads to dose reduction or interruption of treatment such that maximum efficacy cannot be achieved. The most common adverse effects are hyperphosphatemia caused by FGFR1 inhibition and diarrhea due to FGFR4 inhibition, as current therapies are not selective among the FGFRs. Designing selective inhibitors has proved difficult with conventional approaches because the orthosteric sites of FGFR family members are observed to be highly similar in X-ray structures. In this study, aided by analysis of protein dynamics, we designed a selective, covalent FGFR2 inhibitor. In a key initial step, analysis of long-timescale molecular dynamics simulations of the FGFR1 and FGFR2 kinase domains allowed us to identify differential motion in their P-loops, which are located adjacent to the orthosteric site. Using this insight, we were able to design orthosteric binders that selectively and covalently engage the P-loop of FGFR2. Our drug discovery efforts culminated in the development of lirafugratinib (RLY-4008), a covalent inhibitor of FGFR2 that shows substantial selectivity over FGFR1 (~250-fold) and FGFR4 (~5,000-fold) in vitro, causes tumor regression in multiple FGFR2-altered human xenograft models, and was recently demonstrated to be efficacious in the clinic at doses that do not induce clinically significant hyperphosphatemia or diarrhea.

ZNL0325, a Pyrazolopyrimidine-Based Covalent Probe, Demonstrates an Alternative Binding Mode for Kinases

Zhengnian Li, Wenchao Lu, Tyler S. Beyett, Scott B. Ficarro, Jie Jiang, Jason Tse, Audrey Yong-Ju Kim, Jarrod A. Marto, Jianwei Che, Pasi A. Jänne, Michael J. Eck, Tinghu Zhang, and Nathanael S. Gray

Journal of Medicinal Chemistry 2024-5
DOI: 10.1021/acs.jmedchem.3c01891

The pyrazolopyrimidine (PP) heterocycle is a versatile and widely deployed core scaffold for the development of kinase inhibitors. Typically, a 4-amino-substituted pyrazolopyrimidine binds in the ATP-binding pocket in a conformation analogous to the 6-aminopurine of ATP. Here, we report the discovery of ZNL0325 which exhibits a flipped binding mode where the C3 position is oriented toward the ribose binding pocket. ZNL0325 and its analogues feature an acrylamide side chain at the C3 position which is capable of forming a covalent bond with multiple kinases that possess a cysteine at the αD-1 position including BTK, EGFR, BLK, and JAK3. These findings suggest that the ability to form a covalent bond can override the preferred noncovalent binding conformation of the heterocyclic core and provides an opportunity to create structurally distinct covalent kinase inhibitors.