Lindsey Ingerman James

Inhibition of histone trimethyllysine (Kme3) reader proteins is a promising therapeutic strategy due to their roles in gene regulation and disease, particularly cancer. However, developing effective inhibitors has proven challenging because of the extended, solvent-exposed binding interface and the cooperative reliance on interactions with multiple positively charged residues in the histone tail in addition to Kme3. While these cationic groups are often necessary for strong affinity binding, they reduce cell penetration of peptidic inhibitors, increasing the challenge of inhibitor development for this class of proteins. Herein we report the evaluation of three new imidazolium amino acids (IAAs) as novel lipophilic cationic bioisosteres for Kme3, dimethylarginine (Rme2), and Arg against a panel of histone reader proteins. We find that these biomimetics can replace Kme3, Rme2, and Arg, with similar or even improved binding in some cases, while also providing unique selectivity patterns relative to the parent amino acid. Moreover, we find that these imidazolium amino acids promote improved cytosolic penetration due to their increased lipophilicity and lack of hydrogen bond donors. These new lipophilic cationic amino acids provide promising new chemotypes for inhibition of histone reader proteins, as well as other protein–protein interactions that rely on cationic residues.

Inhibition of histone trimethyllysine (Kme3) reader proteins is a promising therapeutic strategy due to their roles in gene regulation and disease, particularly cancer. However, developing effective inhibitors has proven challenging because of the extended, solvent-exposed binding interface and the cooperative reliance on interactions with multiple positively charged residues in the histone tail in addition to Kme3. While these cationic groups are often necessary for strong affinity binding, they reduce cell penetration of peptidic inhibitors, increasing the challenge of inhibitor development for this class of proteins. Herein we report the evaluation of three new imidazolium amino acids as novel lipophilic cationic isosteres for Kme3, dimethylarginine (Rme2), and Arg against a panel of histone reader proteins. We find that these isosteres can replace Kme3, Rme2, and Arg, with equivalent or even improved binding in some cases, while also providing unique selectivity patterns relative to the parent amino acid. Moreover, we find that these imidazolium amino acids promote improved cytosolic penetration due to their increased lipophilicity and lack of hydrogen bond donors. These new isosteres provide promising new chemotypes for inhibition of histone reader proteins, as well as other protein-protein interactions which rely on cationic residues.

Targeted protein degradation (TPD) through the ubiquitin-proteasome system is driven by compound-mediated polyubiquitination of a protein-of-interest by an E3 ubiquitin (Ub) ligase. Relatively few E3s have been successfully utilized for TPD and the governing principles of functional ternary complex formation between the E3, degrader, and protein target remain elusive. FBXO22 has recently been harnessed for TPD applications by degraders that covalently modify its cysteine residues. Here, we reveal that the aldehyde derivative of UNC10088 promotes cooperative binding of FBXO22 to NSD2, a histone methyltransferase and oncogenic protein, leading to a cryo-EM structure of the SKP1-CUL1-F-box (SCF)-FBXO22 complex with NSD2. This structure revealed a conformational change in the FBXO22 loop surrounding C326, further exposing the cysteine for covalent recruitment. Additional medicinal chemistry efforts led to the discovery of benzaldehyde-based non-prodrug degraders that similarly engage C326 of FBXO22 and potently degrade NSD2. Unlike many degraders, our molecules recruit NSD2 to a different surface of FBXO22 than the known FBXO22 substrate BACH1, allowing for concurrent complex formation and structural determination of SCF ^FBXO22 bound to both the neosubstrate NSD2 and native substrate BACH1. Overall, we demonstrate the biochemical and structural basis for NSD2 degradation, revealing key principles for efficient and selective TPD by SCF ^FBXO22 .

PHF1 and PHF19 are key accessory components of polycomb repressive complex 2 (PRC2) which is responsible for trimethylation at histone H3 lysine 27, ultimately leading to gene repression. PHF1 and 19 are known for their methyl-lysine reader Tudor domains that recognize methylated lysine 36 on H3, and they have also been implicated as drivers of a range of cancers, including multiple myeloma. Here, we describe the first small-molecule antagonist of the Tudor domains of PHF1 and 19. We screened an internal library of ∼ 1 K compounds against the Tudor domains using a TR-FRET assay. Upon validation of a hit compound, we performed structure–activity relationship (SAR) studies to better understand the binding mode and improve the affinity of the compound for the PHF1 and 19 Tudor domains. One such compound, UNC7242, which binds PHF1 with a K _d of 1.13 ± 0.04 μM and PHF19 with a K _d of 0.64 ± 0.14 μM, emerged as a lead compound. UNC7242 clearly demonstrates the ligandability of these disease-relevant proteins and serves as a starting point for the future development of potent and selective PHF1 and 19 chemical probes.

Targeted protein degradation (TPD) through the ubiquitin-proteasome system is driven by compound-mediated polyubiquitination of a protein-of-interest by an E3 ubiquitin (Ub) ligase. To date, relatively few E3s have been successfully utilized for TPD and the governing principles of functional ternary complex formation between the E3, degrader, and protein target remain elusive. FBXO22 has recently been harnessed by several groups to target different proteins for degradation. FBXO22 recruitment has been enabled through degraders that covalently modify its cysteine residues. Here, we reveal that the aldehyde derivative of UNC10088 promotes cooperative binding of FBXO22 to NSD2, a histone methyltransferase and oncogenic protein, leading to a cryo-EM structure of the full SKP1-CUL1-F-box (SCF)-FBXO22 complex with NSD2. This structure revealed a conformational change in the FBXO22 loop surrounding C326, further exposing the cysteine for covalent recruitment. Additional medicinal chemistry efforts led to the discovery of benzaldehyde-based non-prodrug degraders that similarly engage C326 of FBXO22 and potently degrade NSD2. Furthermore, unlike many degraders, our molecules recruit NSD2 to a different surface of FBXO22 than the known FBXO22 substrate BACH1, allowing for concurrent complex formation and degradation of both the neosubstrate and endogenous substrates. Overall, we demonstrate the biochemical and structural basis for NSD2 degradation, revealing key principles for efficient and selective TPD by SCF ^FBXO22 .

53BP1 is a DNA damage response protein recruited to sites of double strand breaks through recognition of dimethylated lysine on histone 4 by its tandem Tudor domains. Like 53BP1, BRCA-1 plays a role in the regulation of DNA repair pathways, and BRCA-1 mutations have been strongly linked to breast and ovarian cancer. Interestingly, mice null for 53BP1 and BRCA-1 genes display minimal tumor formation, suggesting that the effects of deleterious BRCA-1 mutations could be prevented with potent 53BP1 small molecule antagonists. Herein, we describe a fragment screen that was used to identify compounds that bind to the 53BP1 Tudor domain and a chemoinformatic workflow to select near-neighbor analogues and establish structure activity relationships for these binders. The marked affinity improvements of the analogues over their parent fragments highlights the developability of these series and the utility of this approach in discovering novel hit compounds for 53BP1 and other methyl-lysine reader proteins.

Ingo Hartung, Lindsey James and Lyn Jones introduce the RSC Medicinal Chemistry themed collection on ‘Induced-Proximity Pharmacology’.

A promising drug target, SETDB1, is a dual methyl-lysine (Kme) reader and methyltransferase implicated in cancer and neurodegenerative disease progression. To help understand the role of the triple Tudor domain (3TD) of SETDB1, its Kme reader, we first identify a low micromolar potency small molecule ligand, UNC6535, which occupies simultaneously both the TD2 and TD3 reader binding sites. Further optimization leads to the discovery of UNC10013, a covalent 3TD ligand targeting Cys385 of SETDB1. UNC10013 is potent with a k_inact/K_I of 1.0 × 10⁶ M⁻¹s⁻¹ and demonstrates proteome-wide selectivity. In cells, negative allosteric modulation of SETDB1-mediated Akt methylation occurs after treatment with UNC10013. Therefore, UNC10013 is a potent, selective, and cell-active covalent ligand for the 3TD of SETDB1, demonstrating negative allosteric modulator properties and making it a promising tool to study the biological role of SETDB1 in disease progression.

Tudor domains are histone readers that can recognize various methylation marks on lysine and arginine. This recognition event plays a key role in the recruitment of other epigenetic effectors and the control of gene accessibility. The Tudor-containing protein family contains 42 members, many of which are involved in the development and progression of various diseases, especially cancer. The development of chemical tools for this family will not only lead to a deeper understanding of the biological functions of Tudor domains but also lay the foundation for therapeutic discoveries. In this review, we discuss the role of several Tudor domain-containing proteins in a range of relevant diseases and progress toward the development of chemical tools such as peptides, peptidomimetics, or small-molecules that bind Tudor domains. Overall, we highlight how Tudor domains are promising targets for therapeutic development and would benefit from the development of novel chemical tools.