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Enzymatic pockets such as those of histone deacetylases (HDACs) are among the most favored targets for drug development. However, enzymatic inhibitors often exhibit low selectivity and high toxicity due to targeting multiple enzyme paralogs, which are often involved in distinct multisubunit complexes. Here, we report the discovery and characterization of a non-enzymatic small molecule inhibitor of HDAC transcriptional repression functions with comparable anti-tumor activity to the enzymatic HDAC inhibitor Vorinostat, and anti-psychedelic activity of anHDAC2knockoutin vivo. We highlight that these phenotypes are achieved while modulating the expression of 20- and 80-fold fewer genes than enzymatic and genetic inhibition in the respective models. Thus, by achieving the same biological outcomes as established therapeutics while impacting a dramatically smaller number of genes, inhibitors of protein-protein interactions can offer important advantages in improving the selectivity of epigenetic modulators.GRAPHICAL ABSTRACT

Summary Identification of selective deubiquitinase (DUB) inhibitors is critical for probe development to further understand and explore DUB biological function. Here, we detail the optimization and deployment of an in vitro fluorogenic ubiquitin-rhodamine assay to conduct high-throughput screening of a small molecule library against a panel of DUBs. In screening the compound library against multiple DUBs in parallel, we describe an approach for identifying selective DUB inhibitors and provide a roadmap for enabling selective DUB inhibitor discovery. For complete details on the use and execution of this protocol, please refer to Varca et al. (2021)., Graphical abstract, Highlights • Expression and purification of deubiquitinase (DUB) enzymes • Buffer screening, miniaturization, and deubiquitinase enzymatic assay optimization • Protocol for high-throughput deubiquitinase inhibitor discovery, Identification of selective deubiquitinase (DUB) inhibitors is critical for probe development to further understand and explore DUB biological function. Here, we detail the optimization and deployment of an in vitro fluorogenic ubiquitin-rhodamine assay to conduct high-throughput screening of a small molecule library against a panel of DUBs. In screening the compound library against multiple DUBs in parallel, we describe an approach for identifying selective DUB inhibitors and provide a roadmap for enabling selective DUB inhibitor discovery.

Deubiquitinating enzymes (DUBs) catalyze the removal of ubiquitin, thereby reversing the activity of E3 ubiquitin ligases and are central to the control of protein abundance and function. Despite the growing interest in DUBs as therapeutic targets, cellular functions for DUBs remain largely unknown and technical challenges often preclude the identification of DUB substrates in a comprehensive manner. Here, we demonstrate that treatment with potent DUB inhibitors coupled to mass spectrometry-based proteomics can identify DUB substrates at a proteome-wide scale. We applied this approach to USP7, a DUB widely investigated as a therapeutic target and identified many known substrates and additional targets. We demonstrate that USP7 substrates are enriched for DNA repair enzymes and E3 ubiquitin ligases. This work provides not only a comprehensive annotation of USP7 substrates, but a general protocol widely applicable to other DUBs, which is critical for translational development of DUB targeted agents.

Deubiquitinating enzymes (DUBs) are a class of isopeptidases that regulate ubiquitin dynamics through catalytic cleavage of ubiquitin from protein substrates and ubiquitin precursors. Despite growing interest in DUB biological function and potential as therapeutic targets, few selective small-molecule inhibitors and no approved drugs currently exist. To identify chemical scaffolds targeting specific DUBs and establish a broader framework for future inhibitor development across the gene family, we performed high-throughput screening of a chemically diverse small-molecule library against eight different DUBs, spanning three well-characterized DUB families. Promising hit compounds were validated in a series of counter-screens and orthogonal assays, as well as further assessed for selectivity across expanded panels of DUBs. Through these efforts, we have identified multiple highly selective DUB inhibitors and developed a roadmap for rapidly identifying and validating selective inhibitors of related enzymes.

NOTCH1 is an attractive cancer target, particularly in T cell acute lymphoblastic leukemia (T-ALL), with activating mutations in this gene identified in more than 50% of cases. In this study, Roti et al. describe the synthesis, characterization, and validation of JQ-FT, a first-in-class NOTCH1 inhibitor that has dual selectivity for leukemia over normal cells and NOTCH1 mutants over wild-type receptors., On-target drug delivery remains a challenge in cancer precision medicine; it is difficult to deliver a targeted therapy to cancer cells without incurring toxicity to normal tissues. The SERCA (sarco-endoplasmic reticulum Ca2+ ATPase) inhibitor thapsigargin inhibits mutant NOTCH1 receptors compared with wild type in T cell acute lymphoblastic leukemia (T-ALL), but its administration is predicted to be toxic in humans. Leveraging the addiction of ALL to folic acid, we conjugated folate to an alcohol derivative of thapsigargin via a cleavable ester linkage. JQ-FT is recognized by folate receptors on the plasma membrane and delivered into leukemia cells as a potent antileukemic agent. In mechanistic and translational models of T-ALL, we demonstrate NOTCH1 inhibition in vitro and in vivo. These proof-of-concept studies support the further optimization of this first-in-class NOTCH1 inhibitor with dual selectivity: leukemia over normal cells and NOTCH1 mutants over wild-type receptors. Furthermore, tumor-specific disruption of Notch signaling may overcome legitimate concerns associated with the tumor suppressor function of nontargeted Notch pathway inhibitors.

The DOT1L lysine methyltransferase has emerged as a validated therapeutic target in MLL-rearranged (MLLr) acute leukemias. Although S-adenosylmethionine competitive inhibitors have demonstrated pharmacological proof-of-principle in MLLr-leukemia, these compounds require further optimization to improve cellular potency and pharmacokinetic stability. Limiting DOT1L inhibitor discovery and ligand optimization have been complex biochemical methods often using radionucleotides and cellular methods requiring prolonged culture. We therefore developed a new suite of assay technologies that allows comparative assessment of chemical tools for DOT1L in a miniaturized format. Coupling these assays with structural information, we developed new insights into DOT1L ligand binding and identified several functionalized probes with increased cellular potency (IC50 values ∼10 nM) and excellent selectivity for DOT1L. Together these assay technologies define a platform capability for discovery and optimization of small-molecule DOT1L inhibitors.

Several studies strongly support the development of Notch1 inhibitors for targeted cancer therapy. This idea is most compelling for T cell acute lymphoblastic leukemia/lymphoma (T-ALL) where activating mutations of NOTCH1 are present in 55-60% of the cases, and cancer dependency has been well established. We previously demonstrated that a small-molecule inhibitor of SERCA (Sarco/Endoplasmic Reticulum Calcium ATPase), thapsigargin, had NOTCH1 dependent anti-leukemia activity in mouse models of human T-ALL. Interestingly thapsigargin preferentially inhibits the maturation of mutant NOTCH1 compared to wild type NOTCH1 and NOTCH2 receptors. This selectivity provides a therapeutic window not observed before with other Notch modulators, including γ-secretase inhibitors or antibody-based approaches. To avoid the potential non-specific toxicityof thapsigargin secondary to intracellular calcium shifts, we leveraged the addiction of ALL to folic acid (FA), and we tagged folate to a permissive site on an active alcohol derivative of thapsigargin (8-O-debutanoylthapsigargin) via a cleavable ester linkage (JQ-FT). We explored folate conjugation of thapsigargin as an anti-cancer strategy for several reasons: the restricted expression of folate receptors (FR1-4) on cancer cells, the affinity of folic acid for FR binding and the FR endocytic mechanism that allows the internalization of large probes. We determined that folate-conjugates, including JQ-FT, enter T-ALL cells by FR2 binding and endocytosis. Importantly, T-ALL cell lines demonstrated stronger folic acid fluorescence-tagged probe (FL-FITC) labeling compared to human CD3+ T cells, providing support for leukemia-specific targeting. To determine if these effects were FR mediated, we treated T-ALL cells with JQ-FT in the presence or absence of the competitive inhibitor FA (10 and 100 μM). Co-treatment with FA diminished the negative growth effects of JQ-FT on T-ALL cells. This result supports FR-mediated internalization of JQ-FT. Next, we demonstrated that JQ-FT alters the growth of T-ALL cell lines, induces apoptosis and a G1/G0 arrest. Similar to thapsigargin, JQ-FT treatment decreased the levels of the activated form of NOTCH1, ICN1, and trans-membrane NOTCH1, while it increased full-length NOTCH1. Consequently, the NOTCH1 transcriptional targets, MYC, DTX1 and HES1, were repressed as measured by qRT-PCR. As predicted, the addition of free FA to the media rescued the decrement of ICN1. To assess the translational significance of these findings, we studied patient-derived xenograft (PDX) cells. JQ-FT treatment inhibited the viability of NOTCH1-mutated PDX cells in vitro. In addition, JQ-FT treatment resulted in a loss of transmembrane NOTCH1, leading to the depletion of ICN1. In contrast, no effect was observed in PDX T-ALL cells possessing wild-type NOTCH1. Consistent with these results, no transcriptional changes were observed in NOTCH1 target genes in wild type PDX samples, supporting the notion that mutated NOTCH1 receptors are more sensitive to SERCA inhibitors, including JQ-FT treatment, than are wild type NOTCH1 receptors in primary patient T-ALL. To explore the therapeutic efficacy of JQ-FT in vivo, we studied its effects on a syngeneic T-ALL mouse model carrying a NOTCH1 L1601P ΔPEST, a common mutation observed in the human disease. We first established the maximal tolerated dose (MTD) of JQ-FT as 60 mg/kg/day in mice administered by daily intraperitoneal injection. Notably, this MTD is 150-fold improved over our prior established MTD of unconjugated thapsigargin. Following five days of treatment, a decrease in tumor growth was observed, confirmed pathologically by a decrease in the leukemic infiltration in the bone marrow, spleen and liver, and clinically by a reduction in spleen weight. Pharmacodynamic modulation of the Notch pathway was validated by measurement of reduced ICN1 expression in T-ALL cells from JQ-FT-treated animals as compared to vehicle-treated controls. In summary, we report the development of the first-in-class NOTCH1 inhibitor with dual selectivity for leukemia over normal cells and NOTCH1-mutant over wild-type receptors. JQ-FT is recognized by folate receptors on the leukemia cell surface and delivered into cells as a mutant NOTCH1-targeted anti-leukemic agent. In mechanistic and translational models of T-ALL, we demonstrated NOTCH1-specific inhibition in vitro and in vivo. Disclosures Bradner: Novartis Institutes for BioMedical Research: Employment.

Cooperation between several epigenetic modulators defines MLL-rearranged leukemia as an epigenomic-driven cancer. Wild type MLL catalyzes trimethylation of lysine 4 on histone 3 from the methyl donor S-adenosylmethionine (SAM) at homeobox and other genes important for hematopoiesis, promoting their expression during development. However, in MLL-rearrangements, its methyltransferase domain is ubiquitously lost and replaced with >70 known fusion partners. Many of these fusion partners recruit DOT1L, the only known SAM-dependent lysine methyltransferase responsible for the methylation of lysine 79 of histone 3 (H3K79)—a mark associated with most actively transcribed genes. Therefore, the recruitment of DOT1L by MLL fusion partners to MLL-target genes leads to aberrant H3K79 hypermethylation at these loci, resulting in inappropriate gene expression and leukemogenesis. DOT1L as a therapeutic target in MLL has been genetically validated by several groups, leading to the development of SAM-competitive small molecule inhibitors of DOT1L. These inhibitors exhibit excellent biochemical activity and selectivity, yet have delayed cellular activity and needing relatively high doses, with viability effects requiring 7-10 days and EC50s for H3K79 methylation depletion of 1-3 μM in cell lines. In animal studies, this translates to a modest survival benefit while requiring high doses through continuous osmotic subcutaneous infusion. Further optimization of DOT1L inhibitors is therefore needed. To date, development of DOT1L inhibitors has been slow, perhaps related to inadequacy of discovery chemistry assay technologies. All biochemical assays are radioactivity-based and are not miniaturizeable; low-throughput and delayed cellular effects of DOT1L inhibition all hamper the discovery of improved inhibitors. Therefore a pressing need towards improved DOT1L inhibitor discovery is a robust, accessible, and rapid profiling platform. Toward this goal, we synthesized both FITC- and biotin-tagged DOT1L probe ligands. We confirmed by structural studies that binding of the probes were similar to our previously published inhibitor, depleted H3K79 methylation, and had antiproliferative effects in MLL-rearranged cell lines. We then utilized the probes to devise two non-radioactive, orthogonal biochemical assays to competitively profile putative inhibitors: one employing bead-based, proxmity fluorescence technology and the second using fluorescence polarization technology. These assays are robust and adaptable to high-throughput screening. We also designed a miniaturizable high-content imaging, immunofluorescence-based assay to assess the effect of DOT1L inhibitors on H3K79 methylation, reporting cellular IC50s after just four days of treatment. These three assays were validated against three known DOT1L inhibitors of different potencies, accurately differentiating between the compounds. Together, these orthogonal assays define an accessible platform capability to discover and optimize DOT1L inhibitors. Our platform rank-ordered a library of SAM derivatives that we synthesized, indicating that large substituents off the SAM base does not affect DOT1L binding. We also explored other features of the SAM core structure, identifying several chlorinated probes that had increased cellular potency (IC50 values ~10nM) relative to the initial compounds published, without losing specificity for DOT1L. The inhibitory effect on MLL-target gene expression correlated to the H3K79me2 decrease reported in high content assay, validating that our high-content assay accurately reports on downstream biology seen later in treatment. And as expected, the high-content potencies of our chlorinated DOT1L probes also correlated to increased anti-proliferative effect in MLL cells. Overall, we utilized chemistry, biology, and chemical biology tools to develop this profiling platform capability for more rapid discovery and optimization of small molecule DOT1L inhibitors. These assays can additionally be used to screen for non-SAM competitive inhibitors in high-throughput fashion. Furthermore, the DOT1L inhibitors and probes synthesized here (available as open-source tools) are useful in deeper mechanistic studies of the DOT1L complex and its role in MLL. Disclosures Armstrong: Epizyme: Consultancy.