Selective Chemical Probes Research Articles

Discovery of CRBN-Dependent WEE1 Molecular Glue Degraders from a Multicomponent Combinatorial Library.

Small molecules promoting protein-protein interactions produce a range of therapeutic outcomes. Molecular glue degraders exemplify this concept due to their compact drug-like structures and ability to engage targets without reliance on existing cognate ligands. While cereblon molecular glue degraders containing glutarimide scaffolds have been approved for treatment of multiple myeloma and acute myeloid leukemia, the design of new therapeutically relevant monovalent degraders remains challenging. We report here an approach to glutarimide-containing molecular glue synthesis using multicomponent reactions as a central modular core-forming step. Screening the resulting library identified HRZ-1 derivatives that target casein kinase 1 α (CK1α) and Wee-like protein kinase (WEE1). Further medicinal chemistry efforts led to identification of selective monovalent WEE1 degraders that provide a potential starting point for the eventual development of a selective chemical degrader probe. The structure of the hit WEE1 degrader complex with CRBN-DDB1 and WEE1 provides a model of the protein-protein interface and ideas to rationalize the observed kinase selectivity. Our findings suggest that modular synthetic routes combined with in-depth structural characterization give access to selective molecular glue degraders and expansion of the CRBN-degradable proteome.

Targeting Poly(U) Binding Splicing Factor 60 (PUF60): A Small-Molecule Inhibitor Shows Anti-Leukemic Activity and Impacts Cell Cycle in Leukemia Models

The emerging role of altered RNA splicing in the development of myeloid neoplasms including MDS and AML has received wide attention. Facilitating genes, including the frequently mutated splicing factors, SF3B1, U2AF1, and SRSF2, have been reported to play a key role in leukemogenesis. Pharmacological intervention targeting cells harboring these mutations has been pursued, but limited agents have been reported. Rare events of more than one hit in splicing factors were found in MDS and AML patients' samples. This suggests that inhibiting another target in the splicing process may overcome the threshold of survival in these vulnerable cells carrying splicing factor mutations and induce toxicity. The concept has been demonstrated in the case of leukemia cells carrying U2AF1 mutations being vulnerable to inhibitors modulating wild-type SF3B1 function. The primary function of U2AF1 is to form a complex with U2AF2 for 3‘ splice site (SS) definition in RNA splicing and lower frequency U2AF2 mutations are also detected in MDS patients, underlying the significance of dysfunctional 3‘ SS definition as a potential facilitator of leukemogenesis. Here, we hypothesize that compounds inhibiting proteins participating in 3' SS selection may disrupt isoform patterns in leukemia cells. These agents may act alone or serve as a second hit in the RNA processing to invoke additional vulnerabilities to leukemia cells carrying the most frequent splicing factor mutations. Definition of 3' SS by U2AF2/U2AF1 is mediated by the recognition of the polypyridine tract (PPT) by U2AF2 and the binding of U2AF1 to the conserved AG dinucleotides at the 3' SS. Like U2AF2, PUF60 contains two zinc-finger domains, recognizing the polyuridine tract preceding 3' SS, and a U2 homology motif (UHM) domain that engages in protein-protein interaction with partner proteins including U2AF2, SF1, and SF3B1. Different from the U2AF1/U2AF2 complex, PUF60 does not have a protein domain to bind to the exon intron boundary junction at the 3' SS. PUF60 has been reported to enhance the splicing activity of U2AF1/U2AF2, but also directly regulates alternative splicing of a subset of genes. A recent finding indicated that knockout of PUF60 leads to alternative splicing switching of CDC25 from isoform A to isoform C to induce cell cycle arrest at G2/M in solid tumors. Leukemia patients carrying PUF60 overexpression have also been found to have less progression free survival (https://ualcan.path.uab.edu/cgi-bin/TCGA-survival1.pl?genenam=PUF60&ctype=LAML) compared to patients with low expression of PUF60. To support our hypothesis, we undertook an inhibitor development campaign to discover small-molecule compounds selectively targeting PUF60. Based on a relatively less selective and weak inhibitor to the UHM domain, SF-153, we conducted chemical modifications and obtained a PUF60 inhibitor, SF2-69, with >15-fold selectivity against U2AF1, RBM39, SPF45, and U2AF2. We found SF2-69 had an IC50 value of ~3 mM in NKM-1 and K562 leukemia cell lines. In the cell cycle analysis, we found SF2-69 increased S and subG1 cell population in NKM-1 at 24h, but induced G1 arrest in K562 at 48h in a dose dependent manner. In comparison, E7820, an RBM39 degrader, increased G2/M phase in both NKM-1 and K562. RNA-seq analysis of NKM-1 treated with SF2-69 after 24h revealed that SF2-69 upregulated a set of genes participating in cholesterol biosynthesis, NME1-NME2 (a tumor suppressor kinase) and downregulated PLK1, a kinase that phosphorylates and activates CDC25C in G2/M transition. Because K562 is p53-null and NKM-1 has wild-type p53, SF2-69 likely induced p53-dependent and p53-independent responses in NKM-1 and K562 cells, respectively. In summary, SF2-69 is a new selective PUF60 inhibitor which disrupts cell cycle progression in leukemia cell lines by modulating p53-mediated responses. Upregulation of cholesterol biosynthesis may be a feedback loop to counteract the inhibition of PUF60 by SF2-69. Further optimization of SF2-69 will allow us to develop more effective and selective chemical probes for studying the role of PUF60 overexpression in leukemia and the potential use of PUF60 inhibitors in MDS and AML cells carrying splicing factor mutations.

Identification and Benchmarking of Myokinasib-II as a Selective and Potent Chemical Probe for Exploring MLCK1 Inhibition.

Deciphering the functional relevance of every protein is crucial to developing a better (patho)physiological understanding of human biology. The discovery and use of quality chemical probes propel exciting developments for developing drugs in therapeutic areas with unmet clinical needs. Myosin light-chain kinase (MLCK) serves as a possible therapeutic target in a plethora of diseases, including inflammatory diseases, cancer, etc. Recent years have seen a substantial increase in interest in exploring MLCK biology. However, there is only one widely used MLCK modulator, namely, ML-7, that too with a narrow working concentration window and high toxicity profile leading to limited insights. Herein, we report the identification of a potent and highly selective chemical probe, Myokinasib-II, from the synthesis and structure-activity relationship studies of a focused indotropane-based compound collection. Notably, it is structurally distinct from ML-7 and hence meets the need for an alternative inhibitor to study MLCK biology as per the recommended best practices. Moreover, our extensive benchmarking studies demonstrate that Myokinasib-II displays better potency, better selectivity profile, and no nonspecific interference in relevant assays as compared to other known MLCK inhibitors.

Understanding the Role of Corrosion and Reactive Oxygen Species in Electrochemical Ozone Production on Nickel and Antimony Doped Tin Oxide

Electrochemical ozone production (EOP), a six-electron reaction, offers promising green applications for water disinfection and organic synthesis. Nickel and antimony-doped tin oxide (Ni/Sb-SnO2, NATO) electrodes are known to be active for EOP. Still, the mechanism of NATO's selectivity and activity and the origin of its instability need to be clarified. We combine electrochemistry, spectroscopic detection of reactive oxygen species (ROS) using selective chemical probes, oxygen anion chemical ionization mass spectrometry (CIMS), and density-functional theory (DFT) simulations to identify the reaction mechanism and investigate the role of corrosion and homogenous reactive oxygen species in EOP on NATO electrodes. We identify hydrogen peroxide as a critical reaction intermediate. Our results suggest that the presence of nickel in NATO enables the catalysis of hydrogen peroxide to hydroperoxyl radicals, which subsequently undergo oxidation to produce ozone. The isotopic makeup of products shows that water is the primary source of ozone, but it also contains oxygen from the metal oxide lattice. The change in the isotopic generation pattern of ozone suggests that NATO catalysts corrode irreversibly, and the lattice oxygen is not regenerated, explaining NATO's instability.

Discovery of Potent and Selective G9a Degraders for the Treatment of Pancreatic Cancer.

G9a, which was initially identified as a histone H3 Lys9 (H3K9) methyltransferase, is potentially an attractive therapeutic target for human cancers. Despite its importance, there is no available selective G9a chemical probe because its homologous protein GLP shares approximately 80% of its sequence with G9a. The development of G9a chemical probes with high selectivity for G9a over GLP is a big challenge but is extremely valuable for understanding G9a-related biology. Herein, we developed a first-in-class selective G9a degrader G9D-4, which induced a dose- and time-dependent G9a degradation without degradation of GLP. G9D-4 exhibited effective antiproliferative activities in a panel of pancreatic cancer cell lines and was able to sensitize KRASG12D mutant pancreatic cancer cells to KRASG12D inhibitor MRTX1133. These data clearly demonstrated the practicality and importance of a selective G9a degrader as a preliminary chemical probe suitable for understanding G9a-related biology and a promising strategy for the treatment of pancreatic cancer.

Primed for Interactions: Investigating the Primed Substrate Channel of the Proteasome for Improved Molecular Engagement.

Protein homeostasis is a tightly conserved process that is regulated through the ubiquitin proteasome system (UPS) in a ubiquitin-independent or ubiquitin-dependent manner. Over the past two decades, the proteasome has become an excellent therapeutic target through inhibition of the catalytic core particle, inhibition of subunits responsible for recognizing and binding ubiquitinated proteins, and more recently, through targeted protein degradation using proteolysis targeting chimeras (PROTACs). The majority of the developed inhibitors of the proteasome's core particle rely on gaining selectivity through binding interactions within the unprimed substrate channel. Although this has allowed for selective inhibitors and chemical probes to be generated for the different proteasome isoforms, much remains unknown about the interactions that could be harnessed within the primed substrate channel to increase potency or selectivity. Herein, we discuss small molecules that interact with the primed substrate pocket and how their differences may give rise to altered activity. Taking advantage of additional interactions with the primed substrate pocket of the proteasome could allow for the generation of improved chemical tools for perturbing or monitoring proteasome activity.

A chemical probe to modulate human GID4 Pro/N-degron interactions.

The C-terminal to LisH (CTLH) complex is a ubiquitin ligase complex that recognizes substrates with Pro/N-degrons via its substrate receptor Glucose-Induced Degradation 4 (GID4), but its function and substrates in humans remain unclear. Here, we report PFI-7, a potent, selective and cell-active chemical probe that antagonizes Pro/N-degron binding to human GID4. Use of PFI-7 in proximity-dependent biotinylation and quantitative proteomics enabled the identification of GID4 interactors and GID4-regulated proteins. GID4 interactors are enriched for nucleolar proteins, including the Pro/N-degron-containing RNA helicases DDX21 and DDX50. We also identified a distinct subset of proteins whose cellular levels are regulated by GID4 including HMGCS1, a Pro/N-degron-containing metabolic enzyme. These data reveal human GID4 Pro/N-degron targets regulated through a combination of degradative and nondegradative functions. Going forward, PFI-7 will be a valuable research tool for investigating CTLH complex biology and facilitating development of targeted protein degradation strategies that highjack CTLH E3 ligase activity.

Gadolinium Doping Modulates the Enzyme-like Activity and Radical-Scavenging Properties of CeO2 Nanoparticles.

Their unique physicochemical properties and multi-enzymatic activity make CeO2 nanoparticles (CeO2 NPs) the most promising active component of the next generation of theranostic drugs. When doped with gadolinium ions, CeO2 NPs constitute a new type of contrast agent for magnetic resonance imaging, possessing improved biocatalytic properties and a high level of biocompatibility. The present study is focused on an in-depth analysis of the enzyme-like properties of gadolinium-doped CeO2 NPs (CeO2:Gd NPs) and their antioxidant activity against superoxide anion radicals, hydrogen peroxide, and alkylperoxyl radicals. Using an anion-exchange method, CeO2:Gd NPs (~5 nm) with various Gd-doping levels (10 mol.% or 20 mol.%) were synthesized. The radical-scavenging properties and biomimetic activities (namely SOD- and peroxidase-like activities) of CeO2:Gd NPs were assessed using a chemiluminescent method with selective chemical probes: luminol, lucigenin, and L-012 (a highly sensitive luminol analogue). In particular, gadolinium doping has been shown to enhance the radical-scavenging properties of CeO2 NPs. Unexpectedly, both bare CeO2 NPs and CeO2:Gd NPs did not exhibit SOD-like activity, acting as pro-oxidants and contributing to the generation of reactive oxygen species. Gadolinium doping caused an increase in the pro-oxidant properties of nanoscale CeO2. At the same time, CeO2:Gd NPs did not significantly inhibit the intrinsic activity of the natural enzyme superoxide dismutase, and CeO2:Gd NPs conjugated with SOD demonstrated SOD-like activity. In contrast to SOD-like properties, peroxidase-like activity was observed for both bare CeO2 NPs and CeO2:Gd NPs. This type of enzyme-like activity was found to be pH-dependent. In a neutral medium (pH = 7.4), nanoscale CeO2 acted as a prooxidant enzyme (peroxidase), while in an alkaline medium (pH = 8.6), it lost its catalytic properties; thus, it cannot be regarded as a nanozyme. Both gadolinium doping and conjugation with a natural enzyme were shown to modulate the interaction of CeO2 NPs with the key components of redox homeostasis.

Investigating impacts of the mycothiazole chemotype as a chemical probe for the study of mitochondrial function and aging.

Small molecule inhibitors of the mitochondrial electron transport chain (ETC) hold significant promise to provide valuable insights to the field of mitochondrial research and aging biology. In this study, we investigated two molecules: mycothiazole (MTZ) - from the marine sponge C. mycofijiensis and its more stable semisynthetic analog 8-O-acetylmycothiazole (8-OAc) as potent and selective chemical probes based on their high efficiency to inhibit ETC complex I function. Similar to rotenone (Rote), MTZ, a newly employed ETC complex I inhibitor, exhibited higher cytotoxicity against cancer cell lines compared to certain non-cancer cell lines. Interestingly, 8-OAc demonstrated greater selectivity for cancer cells when compared to both MTZ and Rote, which has promising potential for anticancer therapeutic development. Furthermore, in vivo experiments with these small molecules utilizing a C. elegans model demonstrate their unexplored potential to investigate aging studies. We observed that both molecules have the ability to induce a mitochondria-specific unfolded protein response (UPRMT) pathway, that extends lifespan of worms when applied in their adult stage. We also found that these two molecules employ different pathways to extend lifespan in worms. Whereas MTZ utilizes the transcription factors ATFS-1 and HSF1, which are involved in the UPRMT and heat shock response (HSR) pathways respectively, 8-OAc only required HSF1 and not ATFS-1 to mediate its effects. This observation underscores the value of applying stable, potent, and selective next generation chemical probes to elucidate an important insight into the functional roles of various protein subunits of ETC complexes and their regulatory mechanisms associated with aging.

Discovery of Inhibitory Fragments That Selectively Target Spire2-FMN2 Interaction.

Here, we report the fragment-based drug discovery of potent and selective fragments that disrupt the Spire2-FMN2 but not the Spire1-FMN2 interaction. Hit fragments were identified in a differential scanning fluorimetry-based screen of an in-house library of 755 compounds and subsequently validated in multiple orthogonal biophysical assays, including fluorescence polarization, microscale thermophoresis, and 1H-15N HSQC nuclear magnetic resonance. Extensive structure-activity relationships combined with molecular docking followed by chemical optimization led to the discovery of compound 13, which exhibits micromolar potency and high ligand efficiency (LE = 0.38). Therefore, this fragment represents a validated starting point for the future development of selective chemical probes targeting the Spire2-FMN2 interaction.

Erratum: Evaluation of a selective chemical probe validates that CK2 mediates neuroinflammation in a human induced pluripotent stem cell-derived microglial model

Erratum: Evaluation of a selective chemical probe validates that CK2 mediates neuroinflammation in a human induced pluripotent stem cell-derived microglial model

Identification of inhibitors targeting the energy-coupling factor (ECF) transporters

The energy-coupling factor (ECF) transporters are a family of transmembrane proteins involved in the uptake of vitamins in a wide range of bacteria. Inhibition of the activity of these proteins could reduce the viability of pathogens that depend on vitamin uptake. The central role of vitamin transport in the metabolism of bacteria and absence from humans make the ECF transporters an attractive target for inhibition with selective chemical probes. Here, we report on the identification of a promising class of inhibitors of the ECF transporters. We used coarse-grained molecular dynamics simulations on Lactobacillus delbrueckii ECF-FolT2 and ECF-PanT to profile the binding mode and mechanism of inhibition of this novel chemotype. The results corroborate the postulated mechanism of transport and pave the way for further drug-discovery efforts.

CK2 Chemical Probes: Past, Present, and Future

Protein kinase casein kinase 2 (CK2/CSNK2) is a pleiotropic kinase involved in many cellular processes and, accordingly, has been identified as a potential target for therapeutic intervention for multiple indications. Significant research effort has been invested into identifying CK2 inhibitors as potential drug candidates and potent and selective CK2 chemical probes to interrogate CK2 function. Here, we review the small molecule inhibitors reported for CK2 and discuss various orthosteric, allosteric, and bivalent inhibitors of CK2. We focus on the pyrazolo[1,5-a]pyrimidines and naphthyridines, two chemotypes that have been extensively explored for chemical probe development. We highlight the uptake and demonstrated utility of the pyrazolo[1,5-a]pyrimidine chemical probe SGC-CK2-1 by the scientific community in cellular studies. Finally, we propose criteria for an ideal in vivo chemical probe for investigating CK2 function in a living organism. While no compound currently meets these metrics, we discuss ongoing and future directions in the development of in vivo chemical probes for CK2.

Chemical proteomics reveals the target landscape of 1,000 kinase inhibitors

Medicinal chemistry has discovered thousands of potent protein and lipid kinase inhibitors. These may be developed into therapeutic drugs or chemical probes to study kinase biology. Because of polypharmacology, a large part of the human kinome currently lacks selective chemical probes. To discover such probes, we profiled 1,183 compounds from drug discovery projects in lysates of cancer cell lines using Kinobeads. The resulting 500,000 compound–target interactions are available in ProteomicsDB and we exemplify how this molecular resource may be used. For instance, the data revealed several hundred reasonably selective compounds for 72 kinases. Cellular assays validated GSK986310C as a candidate SYK (spleen tyrosine kinase) probe and X-ray crystallography uncovered the structural basis for the observed selectivity of the CK2 inhibitor GW869516X. Compounds targeting PKN3 were discovered and phosphoproteomics identified substrates that indicate target engagement in cells. We anticipate that this molecular resource will aid research in drug discovery and chemical biology.

Rational Design and Development of Selective BRD7 Bromodomain Inhibitors and Their Activity in Prostate Cancer.

Bromodomain-containing proteins are readers of acetylated lysine and play important roles in cancer. Bromodomain-containing protein 7 (BRD7) is implicated in multiple malignancies; however, there are no selective chemical probes to study its function in disease. Using crystal structures of BRD7 and BRD9 bromodomains (BDs) bound to BRD9-selective ligands, we identified a binding pocket exclusive to BRD7. We synthesized a series of ligands designed to occupy this binding region and identified two inhibitors with increased selectivity toward BRD7, 1-78 and 2-77, which bind with submicromolar affinity to the BRD7 BD. Our binding mode analyses indicate that these ligands occupy a uniquely accessible binding cleft in BRD7 and maintain key interactions with the asparagine and tyrosine residues critical for acetylated lysine binding. Finally, we validated the utility and selectivity of the compounds in cell-based models of prostate cancer.

Towards the CSNK2 phosphoproteome – With lessons from the COVID-19 pandemic to revealing the secrets of CSNK2 and its promise as a therapeutic target

Dramatic advances in phosphoproteomics and the development of a selective chemical probe have presented new opportunities for revealing the cellular landscape of substrates for CSNK2 (formerly known as CK2 or casein kinase II). In addition to deciphering the role(s) of CSNK2 in physiology and pathophysiology, the CSNK2 phosphoproteome offers the promise of instructing the development of CSNK2-targeted therapy.

Crystal structure of Tudor domain of TDRD3 in complex with a small molecule antagonist

Tudor domain-containing protein 3 (TDRD3) is involved in regulating transcription and translation, promoting breast cancer progression, and modulating neurodevelopment and mental health, making it a promising therapeutic target for associated diseases. The Tudor domain of TDRD3 is essential for its biological functions, and targeting this domain with potent and selective chemical probes may modulate its engagement with chromatin and related functions. Here we reported a study of TDRD3 antagonist following on our earlier work on the development of the SMN antagonist, Compound 1, and demonstrated that TDRD3 can bind effectively to Compound 2, a triple-ring analog of Compound 1. Our structural analysis suggested that the triple-ring compound bound better to TDRD3 due to its smaller side chain at Y566 compared to W102 in SMN. We also revealed that adding a small hydrophobic group to the N-methyl site of Compound 1 can improve binding. These findings provide a path for identifying antagonists for single canonical Tudor domain-containing proteins such as TDRD3 and SMN.

Discovery of a Potent and Selective Naphthyridine-Based Chemical Probe for Casein Kinase 2.

Naphthyridine-based inhibitors were synthesized to yield a potent and cell-active inhibitor of casein kinase 2 (CK2). Compound 2 selectively inhibits CK2α and CK2α' when profiled broadly, thereby making it an exquisitely selective chemical probe for CK2. A negative control that is structurally related but lacks a key hinge-binding nitrogen (7) was designed on the basis of structural studies. Compound 7 does not bind CK2α or CK2α' in cells and demonstrates excellent kinome-wide selectivity. Differential anticancer activity was observed when compound 2 was profiled alongside a structurally distinct CK2 chemical probe: SGC-CK2-1. This naphthyridine-based chemical probe (2) represents one of the best available small molecule tools with which to interrogate biology mediated by CK2.

Proteome-Wide Fragment-Based Ligand and Target Discovery.

Chemical probes are invaluable tools to investigate biological processes and can serve as lead molecules for the development of new therapies. However, despite their utility, only a fraction of human proteins have selective chemical probes, and more generally, our knowledge of the "chemically-tractable" proteome is limited, leaving many potential therapeutic targets unexploited. To help address these challenges, powerful chemical proteomic approaches have recently been developed to globally survey the ability of proteins to bind small molecules (i. e., ligandability) directly in native systems. In this review, we discuss the utility of such approaches, with a focus on the integration of chemoproteomic methods with fragment-based ligand discovery (FBLD), to facilitate the broad mapping of the ligandable proteome while also providing starting points for progression into lead chemical probes.

Accelerating inhibitor discovery for deubiquitinating enzymes

Deubiquitinating enzymes (DUBs) are an emerging drug target class of ~100 proteases that cleave ubiquitin from protein substrates to regulate many cellular processes. A lack of selective chemical probes impedes pharmacologic interrogation of this important gene family. DUBs engage their cognate ligands through a myriad of interactions. We embrace this structural complexity to tailor a chemical diversification strategy for a DUB-focused covalent library. Pairing our library with activity-based protein profiling as a high-density primary screen, we identify selective hits against 23 endogenous DUBs spanning four subfamilies. Optimization of an azetidine hit yields a probe for the understudied DUB VCPIP1 with nanomolar potency and in-family selectivity. Our success in identifying good chemical starting points as well as structure-activity relationships across the gene family from a modest but purpose-build library challenges current paradigms that emphasize ultrahigh throughput in vitro or virtual screens against an ever-increasing scope of chemical space.