Discovery of PFI-6, a small-molecule chemical probe for the YEATS domain of MLLT1 and MLLT3.

Epigenetic proteins containing YEATS domains (YD) are an emerging target class in drug discovery. Described herein are the discovery and characterization efforts associated with PFI-6, a new chemical probe for the YD of MLLT1 (ENL/YEATS1) and MLLT3 (AF9/YEATS3). For hit identification, fragment-like mimetics of endogenous YD ligands (crotonylated histone-containing proteins), were synthesized via parallel medicinal chemistry (PMC) and screened for MLLT1 binding. Subsequent SAR studies led to iterative MLLT1/3 binding and selectivity improvements, culminating in the discovery of PFI-6. PFI-6 demonstrates good affinity and selectivity for MLLT1/3 vs. other human YD proteins (YEATS2/4) and engages MLLT3 in cells. Small-molecule X-ray co-crystal structures of two molecules, including PFI-6, bound to the YD of MLLT1/3 are also described. PFI-6 may be a useful tool molecule to better understand the biological effects associated with modulation of MLLT1/3.

Global Assessment of Drug Target Engagement and Selectivity of Covalent Cysteine-Reactive Inhibitors Using Alkyne-Functionalized Probes.

Covalent inhibitors are emerging as a promising therapeutic means for efficient and sustained targeting of key disease-driving proteins. As for classic non-covalent inhibitors, understanding target engagement and selectivity is essential for determining optimal dosing and limiting potential on- or off-target toxicity. Here, we present a complementary activity-based protein profiling (ABPP) strategy for unbiased proteome-wide profiling of cysteine-reactive inhibitors based on two orthogonal approaches. We illustrate the use of clickable alkyne probes for in-gel fluorescence and mass spectrometry studies using a series of therapeutic XPO1 inhibitors as an example.

Cyclic peptides target the aromatic cage of a PHD-finger reader domain to modulate epigenetic protein function.

Plant homeodomain fingers (PHD-fingers) are a family of reader domains that can recruit epigenetic proteins to specific histone modification sites. Many PHD-fingers recognise methylated lysines on histone tails and play crucial roles in transcriptional regulation, with their dysregulation linked to various human diseases. Despite their biological importance, chemical inhibitors for targeting PHD-fingers are very limited. Here we report a potent and selective de novo cyclic peptide inhibitor (OC9) targeting the Nε-trimethyllysine-binding PHD-fingers of the KDM7 histone demethylases, developed using mRNA display. OC9 disrupts PHD-finger interaction with histone H3K4me3 by engaging the Nε-methyllysine-binding aromatic cage through a valine, revealing a new non-lysine recognition motif for the PHD-fingers that does not require cation-π interaction. PHD-finger inhibition by OC9 impacted JmjC-domain mediated demethylase activity at H3K9me2, leading to inhibition of KDM7B (PHF8) but stimulation of KDM7A (KIAA1718), representing a new approach for selective allosteric modulation of demethylase activity. Chemoproteomic analysis showed selective engagement of OC9 with KDM7s in T cell lymphoblastic lymphoma SUP T1 cells. Our results highlight the utility of mRNA-display derived cyclic peptides for targeting challenging epigenetic reader proteins to probe their biology, and the broader potential of this approach for targeting protein-protein interactions.

Discovery of a Chemical Probe to Study Implications of BPTF Bromodomain Inhibition in Cellular and in†vivo Experiments.

The bromodomain and PHD-finger containing transcription factor (BPTF) is part of the nucleosome remodeling factor (NURF) complex and has been implicated in multiple cancer types. Here, we report the discovery of a potent and selective chemical probe targeting the bromodomain of BPTF with an attractive pharmacokinetic profile enabling cellular and in vivo experiments in mice. Microarray-based transcriptomics in presence of the probe in two lung cancer cell lines revealed only minor effects on the transcriptome. Profiling against a panel of cancer cell lines revealed that the antiproliferative effect does not correlate with BPTF dependency score in depletion screens. Both observations and the multi-domain architecture of BPTF suggest that depleting the protein by proteolysis targeting chimeras (PROTACs) could be a promising strategy to target cancer cell proliferation. We envision that the presented chemical probe and the related negative control will enable the research community to further explore scientific hypotheses with respect to BPTF bromodomain inhibition.

DNA double-strand break-derived RNA drives TIRR/53BP1 complex dissociation.

Tudor-interacting repair regulator (TIRR) is an RNA-binding protein and a negative regulator of the DNA-repair factor p53-binding protein 1 (53BP1). In non-damage conditions, TIRR is bound to 53BP1. After DNA damage, TIRR and 53BP1 dissociate, and 53BP1 binds the chromatin at the double-strand break (DSB) to promote non-homologous end joining (NHEJ)-mediated repair. However, the exact mechanistic details of this dissociation after damage are unknown. Increasing evidence has implicated RNA as a crucial factor in the DNA damage response (DDR). Here, we show that RNA can separate TIRR/53BP1. Specifically, RNA with a hairpin secondary structure, transcribed at the DSB by RNA polymerase II (RNAPII), promotes TIRR/53BP1 complex separation. This hairpin RNA binds to the same residues on TIRR as 53BP1. Our results uncover a role of DNA-damage-derived RNA in modulating a protein-protein interaction and contribute to our understanding of DSB repair.

Chemoproteomic Profiling of Covalent XPO1 Inhibitors to Assess Target Engagement and Selectivity.

Selinexor, a covalent XPO1 inhibitor, is approved in the USA in combination with dexamethasone for penta-refractory multiple myeloma. Additional XPO1 covalent inhibitors are currently in clinical trials for multiple diseases including hematologic malignancies, solid tumor malignancies, glioblastoma multiforme (GBM), and amyotrophic lateral sclerosis (ALS). It is important to measure the target engagement and selectivity of covalent inhibitors to understand the degree of engagement needed for efficacy, while avoiding both mechanism-based and off-target toxicity. Herein, we report clickable probes based on the XPO1 inhibitors selinexor and eltanexor for the labeling of XPO1 in live cells to assess target engagement and selectivity. We used mass spectrometry-based chemoproteomic workflows to profile the proteome-wide selectivity of selinexor and eltanexor and show that they are highly selective for XPO1. Thermal profiling analysis of selinexor further offers an orthogonal approach to measure XPO1 engagement in live cells. We believe these probes and assays will serve as useful tools to further interrogate the biology of XPO1 and its inhibition in cellular and in vivo systems.

Deletion of the deISGylating enzyme USP18 enhances tumour cell antigenicity and radiosensitivity.

BACKGROUND: Interferon (IFN) signalling pathways, a key element of the innate immune response, contribute to resistance to conventional chemotherapy, radiotherapy, and immunotherapy, and are often deregulated in cancer. The deubiquitylating enzyme USP18 is a major negative regulator of the IFN signalling cascade and is the predominant human protease that cleaves ISG15, a ubiquitin-like protein tightly regulated in the context of innate immunity, from its modified substrate proteins in vivo. METHODS: In this study, using advanced proteomic techniques, we have significantly expanded the USP18-dependent ISGylome and proteome in a chronic myeloid leukaemia (CML)-derived cell line. USP18-dependent effects were explored further in CML and colorectal carcinoma cellular models. RESULTS: Novel ISGylation targets were characterised that modulate the sensing of innate ligands, antigen presentation and secretion of cytokines. Consequently, CML USP18-deficient cells are more antigenic, driving increased activation of cytotoxic T lymphocytes (CTLs) and are more susceptible to irradiation. CONCLUSIONS: Our results provide strong evidence for USP18 in regulating antigenicity and radiosensitivity, highlighting its potential as a cancer target.

Re-Evaluating the Mechanism of Action of α,ß-Unsaturated Carbonyl DUB Inhibitors b-AP15 and VLX1570: A Paradigmatic Example of Unspecific Protein Cross-linking with Michael Acceptor Motif-Containing Drugs.

Deubiquitinating enzymes (DUBs) are a growing target class across multiple disease states, with several inhibitors now reported. b-AP15 and VLX1570 are two structurally related USP14/UCH-37 inhibitors. Through a proteomic approach, we demonstrate that these compounds target a diverse range of proteins, resulting in the formation of higher molecular weight (MW) complexes. Activity-based proteome profiling identified CIAPIN1 as a submicromolar covalent target of VLX1570, and further analysis demonstrated that high MW complex formation leads to aggregation of CIAPIN1 in intact cells. Our results suggest that in addition to DUB inhibition, these compounds induce nonspecific protein aggregation, providing molecular explanation for general cellular toxicity.

A Chemical Probe for Tudor Domain Protein Spindlin1 to Investigate Chromatin Function.

Modifications of histone tails, including lysine/arginine methylation, provide the basis of a "chromatin or histone code". Proteins that contain "reader" domains can bind to these modifications and form specific effector complexes, which ultimately mediate chromatin function. The spindlin1 (SPIN1) protein contains three Tudor methyllysine/arginine reader domains and was identified as a putative oncogene and transcriptional coactivator. Here we report a SPIN1 chemical probe inhibitor with low nanomolar in vitro activity, exquisite selectivity on a panel of methyl reader and writer proteins, and with submicromolar cellular activity. X-ray crystallography showed that this Tudor domain chemical probe simultaneously engages Tudor domains 1 and 2 via a bidentate binding mode. Small molecule inhibition and siRNA knockdown of SPIN1, as well as chemoproteomic studies, identified genes which are transcriptionally regulated by SPIN1 in squamous cell carcinoma and suggest that SPIN1 may have a role in cancer related inflammation and/or cancer metastasis.

Fragment-based discovery of a chemical probe for the PWWP1 domain of NSD3.

Here, we report the fragment-based discovery of BI-9321, a potent, selective and cellular active antagonist of the NSD3-PWWP1 domain. The human NSD3 protein is encoded by the WHSC1L1 gene located in the 8p11-p12 amplicon, frequently amplified in breast and squamous lung cancer. Recently, it was demonstrated that the PWWP1 domain of NSD3 is required for the viability of acute myeloid leukemia cells. To further elucidate the relevance of NSD3 in cancer biology, we developed a chemical probe, BI-9321, targeting the methyl-lysine binding site of the PWWP1 domain with sub-micromolar in vitro activity and cellular target engagement at 1 µM. As a single agent, BI-9321 downregulates Myc messenger RNA expression and reduces proliferation in MOLM-13 cells. This first-in-class chemical probe BI-9321, together with the negative control BI-9466, will greatly facilitate the elucidation of the underexplored biological function of PWWP domains.

Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening.

Covalent probes can display unmatched potency, selectivity, and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered nonselective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against 10 cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. In contrast, we found hits for most targets. Combining our approach with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2 and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile-fragment screening as a practical and efficient tool for covalent-ligand discovery.

A chemical biology toolbox to study protein methyltransferases and epigenetic signaling.

Protein methyltransferases (PMTs) comprise a major class of epigenetic regulatory enzymes with therapeutic relevance. Here we present a collection of chemical probes and associated reagents and data to elucidate the function of human and murine PMTs in cellular studies. Our collection provides inhibitors and antagonists that together modulate most of the key regulatory methylation marks on histones H3 and H4, providing an important resource for modulating cellular epigenomes. We describe a comprehensive and comparative characterization of the probe collection with respect to their potency, selectivity, and mode of inhibition. We demonstrate the utility of this collection in CD4+ T cell differentiation assays revealing the potential of individual probes to alter multiple T cell subpopulations which may have implications for T cell-mediated processes such as inflammation and immuno-oncology. In particular, we demonstrate a role for DOT1L in limiting Th1 cell differentiation and maintaining lineage integrity. This chemical probe collection and associated data form a resource for the study of methylation-mediated signaling in epigenetics, inflammation and beyond.

Design, Synthesis and Characterization of Covalent KDM5 Inhibitors.

Histone lysine demethylases (KDMs) are involved in the dynamic regulation of gene expression and they play a critical role in several biological processes. Achieving selectivity over the different KDMs has been a major challenge for KDM inhibitor development. Here we report potent and selective KDM5 covalent inhibitors designed to target cysteine residues only present in the KDM5 sub-family. The covalent binding to the targeted proteins was confirmed by MS and time-dependent inhibition. Additional competition assays show that compounds were non 2-OG competitive. Target engagement and ChIP-seq analysis showed that the compounds inhibited the KDM5 members in cells at nano- to micromolar levels and induce a global increase of the H3K4me3 mark at transcriptional start sites.

Discovery of an MLLT1/3 YEATS Domain Chemical Probe.

YEATS domain (YD) containing proteins are an emerging class of epigenetic targets in drug discovery. Dysregulation of these modified lysine-binding proteins has been linked to the onset and progression of cancers. We herein report the discovery and characterisation of the first small-molecule chemical probe, SGC-iMLLT, for the YD of MLLT1 (ENL/YEATS1) and MLLT3 (AF9/YEATS3). SGC-iMLLT is a potent and selective inhibitor of MLLT1/3-histone interactions. Excellent selectivity over other human YD proteins (YEATS2/4) and bromodomains was observed. Furthermore, our probe displays cellular target engagement of MLLT1 and MLLT3. The first small-molecule X-ray co-crystal structures with the MLLT1 YD are also reported. This first-in-class probe molecule can be used to understand MLLT1/3-associated biology and the therapeutic potential of small-molecule YD inhibitors.

Chemical Instability and Promiscuity of Arylmethylidenepyrazolinone-Based MDMX Inhibitors.

Targeting the protein-protein interaction between p53 and MDM2/MDMX (MDM4) represents an attractive anticancer strategy for the treatment of p53-competent tumors. Several selective and potent MDM2 inhibitors have been developed and entered the clinic; however, the repertoire of MDMX antagonists is still limited. The arylmethylidenepyrazolinone SJ-172550 has been reported as a selective MDMX antagonist; yet, uncertainties about its mechanism of action have raised doubts about its use as a chemical probe. Here, we show that, in addition to its unclear mode of action, SJ-172550 is unstable in aqueous buffers, giving rise to side products of unknown biological activity. Using an SJ-172550-derived affinity probe, we observed promiscuous binding to cellular proteins whereas cellular thermal shift assays did not reveal a stabilizing effect on MDMX. Overall, our results raise further questions about the interpretation of data using SJ-172550 and related compounds to investigate cellular phenotypes.

Small molecule inhibitors reveal an indispensable scaffolding role of RIPK2 in NOD2 signaling.

RIPK2 mediates inflammatory signaling by the bacteria-sensing receptors NOD1 and NOD2. Kinase inhibitors targeting RIPK2 are a proposed strategy to ameliorate NOD-mediated pathologies. Here, we reveal that RIPK2 kinase activity is dispensable for NOD2 inflammatory signaling and show that RIPK2 inhibitors function instead by antagonizing XIAP-binding and XIAP-mediated ubiquitination of RIPK2. We map the XIAP binding site on RIPK2 to the loop between ß2 and ß3 of the N-lobe of the kinase, which is in close proximity to the ATP-binding pocket. Through characterization of a new series of ATP pocket-binding RIPK2 inhibitors, we identify the molecular features that determine their inhibition of both the RIPK2-XIAP interaction, and of cellular and in vivoNOD2 signaling. Our study exemplifies how targeting of the ATP-binding pocket in RIPK2 can be exploited to interfere with the RIPK2-XIAP interaction for modulation of NOD signaling.

Creation of a Novel Class of Potent and Selective MutT Homologue 1 (MTH1) Inhibitors Using Fragment-Based Screening and Structure-Based Drug Design.

Recent literature has both suggested and questioned MTH1 as a novel cancer target. BAY-707 was just published as a target validation small molecule probe for assessing the effects of pharmacological inhibition of MTH1 on tumor cell survival, both in vitro and in vivo. (1) In this report, we describe the medicinal chemistry program creating BAY-707, where fragment-based methods were used to develop a series of highly potent and selective MTH1 inhibitors. Using structure-based drug design and rational medicinal chemistry approaches, the potency was increased over 10,000 times from the fragment starting point while maintaining high ligand efficiency and drug-like properties.

Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement.

For kinase inhibitors, intracellular target selectivity is fundamental to pharmacological mechanism. Although a number of acellular techniques have been developed to measure kinase binding or enzymatic inhibition, such approaches can fail to accurately predict engagement in cells. Here we report the application of an energy transfer technique that enabled the first broad-spectrum, equilibrium-based approach to quantitatively profile target occupancy and compound affinity in live cells. Using this method, we performed a selectivity profiling for clinically relevant kinase inhibitors against 178 full-length kinases, and a mechanistic interrogation of the potency offsets observed between cellular and biochemical analysis. For the multikinase inhibitor crizotinib, our approach accurately predicted cellular potency and revealed improved target selectivity compared with biochemical measurements. Due to cellular ATP, a number of putative crizotinib targets are unexpectedly disengaged in live cells at a clinically relevant drug dose.

Isoform-Selective ATAD2 Chemical Probe with Novel Chemical Structure and Unusual Mode of Action.

ATAD2 (ANCCA) is an epigenetic regulator and transcriptional cofactor, whose overexpression has been linked to the progress of various cancer types. Here, we report a DNA-encoded library screen leading to the discovery of BAY-850, a potent and isoform selective inhibitor that specifically induces ATAD2 bromodomain dimerization and prevents interactions with acetylated histones in vitro, as well as with chromatin in cells. These features qualify BAY-850 as a chemical probe to explore ATAD2 biology.

Lapatinib potentiates cytotoxicity of YM155 in neuroblastoma via inhibition of the ABCB1 efflux transporter.

Adverse side effects of cancer agents are of great concern in the context of childhood tumors where they can reduce the quality of life in young patients and cause life-long adverse effects. Synergistic drug combinations can lessen potential toxic side effects through lower dosing and simultaneously help to overcome drug resistance. Neuroblastoma is the most common cancer in infancy and extremely heterogeneous in clinical presentation and features. Applying a systematic pairwise drug combination screen we observed a highly potent synergy in neuroblastoma cells between the EGFR kinase inhibitor lapatinib and the anticancer compound YM155 that is preserved across several neuroblastoma variants. Mechanistically, the synergy was based on a lapatinib induced inhibition of the multidrug-resistance efflux transporter ABCB1, which is frequently expressed in resistant neuroblastoma cells, which allowed prolonged and elevated cytotoxicity of YM155. In addition, the drug combination (i.e. lapatinib plus YM155) decreased neuroblastoma tumor size in an in vivo model.