A chemical probe targeting the PWWP domain alters NSD2 nucleolar localization.

Nuclear receptor-binding SET domain-containing 2 (NSD2) is the primary enzyme responsible for the dimethylation of lysine 36 of histone 3 (H3K36), a mark associated with active gene transcription and intergenic DNA methylation. In addition to a methyltransferase domain, NSD2 harbors two proline-tryptophan-tryptophan-proline (PWWP) domains and five plant homeodomains (PHDs) believed to serve as chromatin reading modules. Here, we report a chemical probe targeting the N-terminal PWWP (PWWP1) domain of NSD2. UNC6934 occupies the canonical H3K36me2-binding pocket of PWWP1, antagonizes PWWP1 interaction with nucleosomal H3K36me2 and selectively engages endogenous NSD2 in cells. UNC6934 induces accumulation of endogenous NSD2 in the nucleolus, phenocopying the localization defects of NSD2 protein isoforms lacking PWWP1 that result from translocations prevalent in multiple myeloma (MM). Mutations of other NSD2 chromatin reader domains also increase NSD2 nucleolar localization and enhance the effect of UNC6934. This chemical probe and the accompanying negative control UNC7145 will be useful tools in defining NSD2 biology.

Assessing the performance of docking, FEP, and MM/GBSA methods on a series of KLK6 inhibitors.

Kallikrein 6 (KLK6) is an attractive drug target for the treatment of neurological diseases and for various cancers. Herein, we explore the accuracy and efficiency of different computational methods and protocols to predict the free energy of binding (ΔGbind) for a series of 49 inhibitors of KLK6. We found that the performance of the methods varied strongly with the tested system. For only one of the three KLK6 datasets, the docking scores obtained with rDock were in good agreement (R2 ≥ 0.5) with experimental values of ΔGbind. A similar result was obtained with MM/GBSA (using the ff14SB force field) calculations based on single minimized structures. Improved binding affinity predictions were obtained with the free energy perturbation (FEP) method, with an overall MUE and RMSE of 0.53 and 0.68 kcal/mol, respectively. Furthermore, in a simulation of a real-world drug discovery project, FEP was able to rank the most potent compounds at the top of the list. These results indicate that FEP can be a promising tool for the structure-based optimization of KLK6 inhibitors.

Methyltransferase Inhibitors: Competing with, or Exploiting the Bound Cofactor.

Protein methyltransferases (PMTs) are enzymes involved in epigenetic mechanisms, DNA repair, and other cellular machineries critical to cellular identity and function, and are an important target class in chemical biology and drug discovery. Central to the enzymatic reaction is the transfer of a methyl group from the cofactor S-adenosylmethionine (SAM) to a substrate protein. Here we review how the essentiality of SAM for catalysis is exploited by chemical inhibitors. Occupying the cofactor binding pocket to compete with SAM can be hindered by the hydrophilic nature of this site, but structural studies of compounds now in the clinic revealed that inhibitors could either occupy juxtaposed pockets to overlap minimally, but sufficiently with the bound cofactor, or induce large conformational remodeling leading to a more druggable binding site. Rather than competing with the cofactor, other inhibitors compete with the substrate and rely on bound SAM, either to allosterically stabilize the substrate binding site, or for direct SAM-inhibitor interactions.

Discovery and Characterization of a Chemical Probe Targeting the Zinc-Finger Ubiquitin-Binding Domain of HDAC6.

Histone deacetylase 6 (HDAC6) inhibition is an attractive strategy for treating numerous cancers, and HDAC6 catalytic inhibitors are currently in clinical trials. The HDAC6 zinc-finger ubiquitin-binding domain (UBD) binds free C-terminal diglycine motifs of unanchored ubiquitin polymer chains and protein aggregates, playing an important role in autophagy and aggresome assembly. However, targeting this domain with small molecule antagonists remains an underdeveloped avenue of HDAC6-focused drug discovery. We report SGC-UBD253 (25), a chemical probe potently targeting HDAC6-UBD in vitro with selectivity over nine other UBDs, except for weak USP16 binding. In cells, 25 is an effective antagonist of HDAC6-UBD at 1 µM, with marked proteome-wide selectivity. We identified SGC-UBD253N (32), a methylated derivative of 25 that is 300-fold less active, serving as a negative control. Together, 25 and 32 could enable further exploration of the biological function of the HDAC6-UBD and investigation of the therapeutic potential of targeting this domain.

Discovery of Small-Molecule Antagonists of the PWWP Domain of NSD2.

Increased activity of the lysine methyltransferase NSD2 driven by translocation and activating mutations is associated with multiple myeloma and acute lymphoblastic leukemia, but no NSD2-targeting chemical probe has been reported to date. Here, we present the first antagonists that block the protein-protein interaction between the N-terminal PWWP domain of NSD2 and H3K36me2. Using virtual screening and experimental validation, we identified the small-molecule antagonist 3f, which binds to the NSD2-PWWP1 domain with a Kd of 3.4 µM and abrogates histone H3K36me2 binding to the PWWP1 domain in cells. This study establishes an alternative approach to targeting NSD2 and provides a small-molecule antagonist that can be further optimized into a chemical probe to better understand the cellular function of this protein.

Selective, Small-Molecule Co-Factor Binding Site Inhibition of a Su(var)3-9, Enhancer of Zeste, Trithorax Domain Containing Lysine Methyltransferase.

The first chemical probe to primarily occupy the co-factor binding site of a Su(var)3-9, enhancer of a zeste, trithorax (SET) domain containing protein lysine methyltransferase (PKMT) is reported. Protein methyltransferases require S-adenosylmethionine (SAM) as a co-factor (methyl donor) for enzymatic activity. However, SAM itself represents a poor medicinal chemistry starting point for a selective, cell-active inhibitor given its extreme physicochemical properties and its role in multiple cellular processes. A previously untested medicinal chemistry strategy of deliberate file enrichment around molecules bearing the hallmarks of SAM, but with improved lead-like properties from the outset, yielded viable hits against SET and MYND domain-containing protein 2 (SMYD2) that were shown to bind in the co-factor site. These leads were optimized to identify a highly biochemically potent, PKMT-selective, and cell-active chemical probe. While substrate-based inhibitors of PKMTs are known, this represents a novel, co-factor-derived strategy for the inhibition of SMYD2 which may also prove applicable to lysine methyltransferase family members previously thought of as intractable.

Identification and Structure-Activity Relationship of HDAC6 Zinc-Finger Ubiquitin Binding Domain Inhibitors.

HDAC6 plays a central role in the recruitment of protein aggregates for lysosomal degradation and is a promising target for combination therapy with proteasome inhibitors in multiple myeloma. Pharmacologically displacing ubiquitin from the zinc-finger ubiquitin-binding domain (ZnF-UBD) of HDAC6 is an underexplored alternative to catalytic inhibition. Here, we present the discovery of an HDAC6 ZnF-UBD-focused chemical series and its progression from virtual screening hits to low micromolar inhibitors. A carboxylate mimicking the C-terminal extremity of ubiquitin, and an extended aromatic system stacking with W1182 and R1155, are necessary for activity. One of the compounds induced a conformational remodeling of the binding site where the primary binding pocket opens up onto a ligand-able secondary pocket that may be exploited to increase potency. The preliminary structure-activity relationship accompanied by nine crystal structures should enable further optimization into a chemical probe to investigate the merit of targeting the ZnF-UBD of HDAC6 in multiple myeloma and other diseases.

A systematic analysis of atomic protein-ligand interactions in the PDB.

As the protein databank (PDB) recently passed the cap of 123 456 structures, it stands more than ever as an important resource not only to analyze structural features of specific biological systems, but also to study the prevalence of structural patterns observed in a large body of unrelated structures, that may reflect rules governing protein folding or molecular recognition. Here, we compiled a list of 11 016 unique structures of small-molecule ligands bound to proteins - 6444 of which have experimental binding affinity - representing 750 873 protein-ligand atomic interactions, and analyzed the frequency, geometry and impact of each interaction type. We find that hydrophobic interactions are generally enriched in high-efficiency ligands, but polar interactions are over-represented in fragment inhibitors. While most observations extracted from the PDB will be familiar to seasoned medicinal chemists, less expected findings, such as the high number of C-H···O hydrogen bonds or the relatively frequent amide-π stacking between the backbone amide of proteins and aromatic rings of ligands, uncover underused ligand design strategies.

Small Molecule Antagonists of the Interaction between the Histone Deacetylase 6 Zinc-Finger Domain and Ubiquitin.

Inhibitors of HDAC6 have attractive potential in numerous cancers. HDAC6 inhibitors to date target the catalytic domains, but targeting the unique zinc-finger ubiquitin-binding domain (Zf-UBD) of HDAC6 may be an attractive alternative strategy. We developed X-ray crystallography and biophysical assays to identify and characterize small molecules capable of binding to the Zf-UBD and competing with ubiquitin binding. Our results revealed two adjacent ligand-able pockets of HDAC6 Zf-UBD and the first functional ligands for this domain.

Discovery of a Potent and Selective Coactivator Associated Arginine Methyltransferase 1 (CARM1) Inhibitor by Virtual Screening.

Protein arginine methyltransferases (PRMTs) represent an emerging target class in oncology and other disease areas. So far, the most successful strategy to identify PRMT inhibitors has been to screen large to medium-size chemical libraries. Attempts to develop PRMT inhibitors using receptor-based computational methods have met limited success. Here, using virtual screening approaches, we identify 11 CARM1 (PRMT4) inhibitors with ligand efficiencies ranging from 0.28 to 0.84. CARM1 selective hits were further validated by orthogonal methods. Two structure-based rounds of optimization produced 27 (SGC2085), a CARM1 inhibitor with an IC50 of 50 nM and more than hundred-fold selectivity over other PRMTs. These results indicate that virtual screening strategies can be successfully applied to Rossmann-fold protein methyltransferases.

Discovery of a Potent Class I Protein Arginine Methyltransferase Fragment Inhibitor.

Protein methyltransferases (PMTs) are a promising target class in oncology and other disease areas. They are composed of SET domain methyltransferases and structurally unrelated Rossman-fold enzymes that include protein arginine methyltransferases (PRMTs). In the absence of a well-defined medicinal chemistry tool-kit focused on PMTs, most current inhibitors were identified by screening large and diverse libraries of leadlike molecules. So far, no successful fragment-based approach was reported against this target class. Here, by deconstructing potent PRMT inhibitors, we find that chemical moieties occupying the substrate arginine-binding site can act as efficient fragment inhibitors. Screening a fragment library against PRMT6 produced numerous hits, including a 300 nM inhibitor (ligand efficiency of 0.56) that decreased global histone 3 arginine 2 methylation in cells, and can serve as a warhead for the development of PRMT chemical probes.

Structural biology and chemistry of protein arginine methyltransferases.

Protein arginine methyltransferases (PRMTs), an emerging target class in drug discovery, can methylate histones and other substrates, and can be divided into three subgroups, based on the methylation pattern of the reaction product (monomethylation, symmetrical or asymmetrical dimethylation). Here, we review the growing body of structural information characterizing this protein family, including structures in complex with substrate-competitive and allosteric inhibitors. We outline structural differences between type I, II and III enzymes and propose a model underlying class-specificity. We analyze the structural plasticity and diversity of the substrate, cofactor and allosteric binding sites, and propose that the conformational dynamics of PRMTs can be exploited towards the discovery of allosteric inhibitors that would antagonize conformationally active states.