Recruitment of FBXO22 for Targeted Degradation of NSD2.

Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here, we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain of function mutation p.E1099K, resulting in growth suppression, apoptosis, and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 in a covalent and reversible manner to recruit the SCF FBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCF FBXO22 . Overall, we present a highly potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a novel FBXO22-dependent TPD strategy.

PROTAC Linkerology Leads to an Optimized Bivalent Chemical Degrader of Polycomb Repressive Complex 2 (PRC2) Components.

Bivalent chemical degraders, otherwise known as proteolysis-targeting chimeras (PROTACs), have proven to be an efficient strategy for targeting overexpressed or mutated proteins in cancer. PROTACs provide an alternative approach to small-molecule inhibitors, which are restricted by occupancy-driven pharmacology, often resulting in acquired inhibitor resistance via compensatory increases in protein expression. Despite the advantages of bivalent chemical degraders, they often have suboptimal physicochemical properties and optimization for efficient degradation remains highly unpredictable. Herein, we report the development of a potent EED-targeted PRC2 degrader, UNC7700. UNC7700 contains a unique cis-cyclobutane linker and potently degrades PRC2 components EED (DC50 = 111 nM; Dmax = 84%), EZH2WT/EZH2Y641N (DC50 = 275 nM; Dmax = 86%), and to a lesser extent SUZ12 (Dmax = 44%) after 24 h in a diffuse large B-cell lymphoma DB cell line. Characterization of UNC7700 and related compounds for ternary complex formation and cellular permeability to provide a rationale for the observed improvement in degradation efficiency remained challenging. Importantly, UNC7700 dramatically reduces H3K27me3 levels and is anti-proliferative in DB cells (EC50 = 0.79 ± 0.53 µM).

Discovery of a Potent and Selective Targeted NSD2 Degrader for the Reduction of H3K36me2.

Nuclear receptor-binding SET domain-containing 2 (NSD2) plays important roles in gene regulation, largely through its ability to dimethylate lysine 36 of histone 3 (H3K36me2). Despite aberrant activity of NSD2 reported in numerous cancers, efforts to selectively inhibit the catalytic activity of this protein with small molecules have been unsuccessful to date. Here, we report the development of UNC8153, a novel NSD2-targeted degrader that potently and selectively reduces the cellular levels of both NSD2 protein and the H3K36me2 chromatin mark. UNC8153 contains a simple warhead that confers proteasome-dependent degradation of NSD2 through a novel mechanism. Importantly, UNC8153-mediated reduction of H3K36me2 through the degradation of NSD2 results in the downregulation of pathological phenotypes in multiple myeloma cells including mild antiproliferative effects in MM1.S cells containing an activating point mutation and antiadhesive effects in KMS11 cells harboring the t(4;14) translocation that upregulates NSD2 expression.

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.

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.

Targeting Regorafenib-Induced Toxicity through Inhibition of Gut Microbial ß-Glucuronidases.

Regorafenib (Stivarga) is an oral small molecule kinase inhibitor used to treat metastatic colorectal cancer, hepatocellular carcinomas, and gastrointestinal stromal tumors. Diarrhea is one of the most frequently observed adverse reactions associated with regorafenib. This toxicity may arise from the reactivation of the inactive regorafenib-glucuronide to regorafenib by gut microbial ß-glucuronidase (GUS) enzymes in the gastrointestinal tract. We sought to unravel the molecular basis of regorafenib-glucuronide processing by human intestinal GUS enzymes and to examine the potential inhibition of these enzymes. Using a panel of 31 unique gut microbial GUS enzymes derived from the 279 mapped from the human gut microbiome, we found that only four were capable of regorafenib-glucuronide processing. Using crystal structures as a guide, we pinpointed the molecular features unique to these enzymes that confer regorafenib-glucuronide processing activity. Furthermore, a pilot screen identified the FDA-approved drug raloxifene as an inhibitor of regorafenib reactivation by the GUS proteins discovered. Novel synthetic raloxifene analogs exhibited improved potency in both in vitro and ex vivo studies. Taken together, these data establish that regorafenib reactivation is exclusively catalyzed by gut microbial enzymes and that these enzymes are amenable to targeted inhibition. Our results unravel key molecular details of regorafenib reactivation in the GI tract and provide a potential pathway to improve clinical outcomes with regorafenib.

Effects of Isx-9 and stress on adult hippocampal neurogenesis: Experimental considerations and future perspectives.

The development of synthetic small molecules capable of promoting neuronal fate in stem cells is a promising strategy to prevent the decline of hippocampal function caused by several neurological disorders. Within this context, isoxazole 9 (Isx-9) has been shown to strongly induce cell proliferation and neuronal differentiation in the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG), while also improving hippocampal function in healthy mice. We have recently demonstrated that Isx-9 is able to restore normal neurogenesis levels after procedural stress. Here, we further discuss these findings highlighting the importance of including a naïve group in studies investigating the effects of either restraint stress or mild chronic unpredictable stress (CUS) on adult hippocampal neurogenesis.

ISX-9 can potentiate cell proliferation and neuronal commitment in the rat dentate gyrus.

Adult hippocampal neurogenesis can be modulated by various physiological and pathological conditions, including stress, affective disorders, and several neurological conditions. Given the proposed role of this form of structural plasticity in the functioning of the hippocampus (namely learning and memory and affective behaviors), it is believed that alterations in hippocampal neurogenesis might underlie some of the behavioral deficits associated with these psychiatric and neurological conditions. Thus, the search for compounds that can reverse these deficits with minimal side effects has become a recognized priority. In the present study we tested the pro-neurogenic effects of isoxazole 9 (Isx-9), a small synthetic molecule that has been recently identified through the screening of chemical libraries in stem cell-based assays. We found that administration of Isx-9 for 14days was able to potentiate cell proliferation and increase the number of immature neurons in the hippocampal DG of adult rats. In addition, Isx-9 treatment was able to completely reverse the marked reduction in these initial stages of the neurogenic process observed in vehicle-treated animals (which were submitted to repeated handling and exposure to daily intraperitoneal injections). Based on these results, we recommend that future neurogenesis studies that require repeated handling and manipulation of animals should include a naïve (non-manipulated) control to determine the baseline levels of hippocampal cell proliferation and neuronal differentiation. Overall, these findings demonstrate that Isx-9 is a promising synthetic compound for the mitigation of stress-induced deficits in adult hippocampal neurogenesis. Future studies are thus warranted to evaluate the pro-neurogenic properties of Isx-9 in animal models of affective and neurological disorders associated with impaired hippocampal structural plasticity.

Salicylates are interference compounds in TR-FRET assays.

Given the importance of high-throughput screening in drug discovery, the identification of compounds that interfere with assay readouts is crucial. The pursuit of false positives wastes time and money, while distracting development teams from more promising leads. In the context of TR-FRET assays, most interfering compounds are dyes or aggregators. In the course of our studies on the PD1-PDL2 interaction, we discovered that salicylic acids, an extremely common compound subclass in screening libraries, interfere with TR-FRET assays. While the precise mechanism of interference was not established, our data suggest that interaction of the salicylate with the cryptand-ligated europium FRET donor is responsible for the change in assay signal.