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.

Exploiting the Cullin E3 Ligase Adaptor Protein SKP1 for Targeted Protein Degradation

Seong Ho Hong, Anand Divakaran, Akane Osa, Oscar W. Huang, Ingrid E. Wertz, and Daniel K. Nomura

ACS Chemical Biology 2024

DOI: https://doi.org/10.1021/acschembio.3c0064

Targeted protein degradation with proteolysis targeting chimeras (PROTACs) is a powerful therapeutic modality for eliminating disease-causing proteins through targeted ubiquitination and proteasome-mediated degradation. Most PROTACs have exploited substrate receptors of Cullin-RING E3 ubiquitin ligases such as cereblon and VHL. Whether core, shared, and essential components of the Cullin-RING E3 ubiquitin ligase complex can be used for PROTAC applications remains less explored. Here, we discovered a cysteine-reactive covalent recruiter EN884 against the SKP1 adapter protein of the SKP1-CUL1-F-box containing the SCF complex. We further showed that this recruiter can be used in PROTAC applications to degrade neo-substrate proteins such as BRD4 and the androgen receptor in a SKP1- and proteasome-dependent manner. Our studies demonstrate that core and essential adapter proteins within the Cullin-RING E3 ubiquitin ligase complex can be exploited for targeted protein degradation applications and that covalent chemoproteomic strategies can enable recruiter discovery against these targets.

Kinase-impaired BTK mutations are susceptible to clinical-stage BTK and IKZF1/3 degrader NX-2127

• Skye Montoya et al.
 

Science383,eadi5798(2024).

 DOI: 10.1126/science.adi5798

Structured Abstract

INTRODUCTION

Bruton’s tyrosine kinase (BTK) is a nonreceptor kinase in the B cell receptor (BCR) signaling cascade critical for B cell survival. As such, chronic lymphocytic leukemia (CLL) and other B cell cancers are sensitive to inhibition of BTK. Covalent and noncovalent inhibitors of BTK have revolutionized the treatment of these cancers. Therefore, understanding mechanisms by which acquired mutations in BTK confer drug resistance and developing new therapies to overcome resistance are critically important.

RATIONALE

We recently discovered BTK mutations that confer resistance across covalent and noncovalent BTK inhibitors. In this study, we found that a group of these mutants impair BTK kinase activity despite still enabling downstream BCR signaling. We therefore set out to understand the nonenzymatic functions of BTK and explored targeted protein degradation to overcome the oncogenic scaffold function of mutant BTK. This effort included evaluation of BTK degradation in patients with CLL treated in a phase 1 clinical trial of NX-2127, a first-in-class BTK degrader (NCT04830137).

RESULTS

BTK enzymatic activity assays revealed that drug resistance mutations in BTK fall into two distinct groups: kinase proficient and kinase impaired. Immunoprecipitation mass spectrometry of kinase-impaired BTK L528W (Leu528→Trp) revealed a scaffold function of BTK with downstream signaling and survival dependent on surrogate kinases that bind to kinase-impaired BTK proteoforms. To target the nonenzymatic functions of BTK, we developed NX-2127, a heterobifunctional molecule that engages the ubiquitin-proteasome system to simultaneously bind both BTK and the cereblon E3 ubiquitin ligase complex, inducing polyubiquitination and proteasome-dependent degradation of IKZF1/3 and all recurrent drug-resistant forms of mutant BTK. The activity of NX-2127 on BTK degradation was further demonstrated in patients with CLL treated in a phase 1 clinical trial of NX-2127, where >80% BTK degradation was achieved and clinical responses were also seen in 79% of evaluable patients, independent of mutant BTK genotypes.

CONCLUSION

We identified that BTK inhibitor resistance mutations fall into two distinct functional categories. Kinase-impaired BTK mutants disable BTK kinase activity while promoting physical interactions with other kinases to sustain downstream BCR signaling. This scaffold function of BTK was disrupted by NX-2127, a potent BTK degrader, which showed promising responses for patients with relapsed and refractory CLL, independently of mutant BTK functional category.

Location-agnostic site-specific protein bioconjugation via Baylis Hillman adducts

Mir, M.H., Parmar, S., Singh, C. et al.

 Nat Commun 15, 859 (2024). 

https://doi.org/10.1038/s41467-024-45124-2

Proteins labelled site-specifically with small molecules are valuable assets for chemical biology and drug development. The unique reactivity profile of the 1,2-aminothiol moiety of N-terminal cysteines (N-Cys) of proteins renders it highly attractive for regioselective protein labelling. Herein, we report an ultrafast Z-selective reaction between isatin-derived Baylis Hillman adducts and 1,2-aminothiols to form a bis-heterocyclic scaffold, and employ it for stable protein bioconjugation under both in vitro and live-cell conditions. We refer to our protein bioconjugation technology as Baylis Hillman orchestrated protein aminothiol labelling (BHoPAL). Furthermore, we report a lipoic acid ligase-based technology for introducing the 1,2-aminothiol moiety at any desired site within proteins, rendering BHoPAL location-agnostic (not limited to N-Cys). By using this approach in tandem with BHoPAL, we generate dually labelled protein bioconjugates appended with different labels at two distinct specific sites on a single protein molecule. Taken together, the protein bioconjugation toolkit that we disclose herein will contribute towards the generation of both mono and multi-labelled protein-small molecule bioconjugates for applications as diverse as biophysical assays, cellular imaging, and the production of therapeutic protein–drug conjugates. In addition to protein bioconjugation, the bis-heterocyclic scaffold we report herein will find applications in synthetic and medicinal chemistry.