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.

Improvement of Aqueous Solubility of Lapatinib-Derived Analogues: Identification of a Quinolinimine Lead for Human African Trypanosomiasis Drug Development.

Lapatinib, an approved epidermal growth factor receptor inhibitor, was explored as a starting point for the synthesis of new hits against Trypanosoma brucei, the causative agent of human African trypanosomiasis (HAT). Previous work culminated in 1 (NEU-1953), which was part of a series typically associated with poor aqueous solubility. In this report, we present various medicinal chemistry strategies that were used to increase the aqueous solubility and improve the physicochemical profile without sacrificing antitrypanosomal potency. To rank trypanocidal hits, a new assay (summarized in a cytocidal effective concentration (CEC50)) was established, as part of the lead selection process. Increasing the sp3 carbon content of 1 resulted in 10e (0.19 µM EC50 against T. brucei and 990 µM aqueous solubility). Further chemical exploration of 10e yielded 22a, a trypanocidal quinolinimine (EC50: 0.013 µM; aqueous solubility: 880 µM; and CEC50: 0.18 µM). Compound 22a reduced parasitemia 109 fold in trypanosome-infected mice; it is an advanced lead for HAT drug development.

Anilinoquinoline based inhibitors of trypanosomatid proliferation.

We recently reported the medicinal chemistry re-optimization of a series of compounds derived from the human tyrosine kinase inhibitor, lapatinib, for activity against Plasmodium falciparum. From this same library of compounds, we now report potent compounds against Trypanosoma brucei brucei (which causes human African trypanosomiasis), T. cruzi (the pathogen that causes Chagas disease), and Leishmania spp. (which cause leishmaniasis). In addition, sub-micromolar compounds were identified that inhibit proliferation of the parasites that cause African animal trypanosomiasis, T. congolense and T. vivax. We have found that this set of compounds display acceptable physicochemical properties and represent progress towards identification of lead compounds to combat several neglected tropical diseases.

Gut Microbial ß-Glucuronidase Inhibition via Catalytic Cycle Interception.

Microbial ß-glucuronidases (GUSs) cause severe gut toxicities that limit the efficacy of cancer drugs and other therapeutics. Selective inhibitors of bacterial GUS have been shown to alleviate these side effects. Using structural and chemical biology, mass spectrometry, and cell-based assays, we establish that piperazine-containing GUS inhibitors intercept the glycosyl-enzyme catalytic intermediate of these retaining glycosyl hydrolases. We demonstrate that piperazine-based compounds are substrate-dependent GUS inhibitors that bind to the GUS-GlcA catalytic intermediate as a piperazine-linked glucuronide (GlcA, glucuronic acid). We confirm the GUS-dependent formation of inhibitor-glucuronide conjugates by LC-MS and show that methylated piperazine analogs display significantly reduced potencies. We further demonstrate that a range of approved piperazine- and piperidine-containing drugs from many classes, including those for the treatment of depression, infection, and cancer, function by the same mechanism, and we confirm through gene editing that these compounds selectively inhibit GUS in living bacterial cells. Together, these data reveal a unique mechanism of GUS inhibition and show that a range of therapeutics may impact GUS activities in the human gut.

Optimization of Physicochemical Properties for 4-Anilinoquinoline Inhibitors of Plasmodium falciparum Proliferation.

We recently reported the medicinal chemistry reoptimization of a known human tyrosine kinase inhibitor, lapatinib, against a variety of parasites responsible for numerous tropical diseases, including human African trypanosomiasis ( Trypanosoma brucei), Chagas disease ( T. cruzi), Leishmaniasis ( Leishmania spp.), and malaria ( Plasmodium falciparum). Herein, we report our continuing efforts to optimize this series against P. falciparum. Through the design of a library of compounds focused on reducing the lipophilicity and molecular weight, followed by an SAR exploration, we have identified NEU-1953 (40). This compound is a potent inhibitor of P. falciparum with an improved ADME profile over the previously reported compound, NEU-961 (3).