Targeting the “undruggable” proteome remains one of the big challenges in drug discovery. Recent innovations in the field of targeted protein degradation and manipulation of the ubiquitin-proteasome ...system open up new therapeutic approaches for disorders that cannot be targeted with conventional inhibitor paradigms. Proteolysis targeting chimeras (PROTACs) are bivalent ligands in which a compound that binds to the protein target of interest is connected to a second molecule that binds an E3 ligase via a linker. The E3 protein is usually either Cereblon or Von Hippel-Lindau. Several examples of selective PROTAC molecules with potent effect in cells and in vivo models have been reported. The degradation of specific proteins via these bivalent molecules is already allowing for the study of biochemical pathways and cell biology with more specificity than was possible with inhibitor compounds. In this review, we provide a comprehensive overview of recent developments in the field of small molecule mediated protein degradation, including transcription factors, kinases and nuclear receptors. We discuss the potential benefits of protein degradation over inhibition as well as the challenges that need to be overcome.
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Designed Spiroketal Protein Modulation Scheepstra, Marcel; Andrei, Sebastian A.; Unver, M. Yagiz ...
Angewandte Chemie,
May 8, 2017, Letnik:
56, Številka:
20
Journal Article
Recenzirano
Odprti dostop
Spiroketals are structural motifs found in many biologically active natural products, which has stimulated considerable efforts toward their synthesis and interest in their use as drug lead ...compounds. Despite this, the use of spiroketals, and especially bisbenzanulated spiroketals, in a structure‐based drug discovery setting has not been convincingly demonstrated. Herein, we report the rational design of a bisbenzannulated spiroketal that potently binds to the retinoid X receptor (RXR) thereby inducing partial co‐activator recruitment. We solved the crystal structure of the spiroketal–hRXRα–TIF2 ternary complex, and identified a canonical allosteric mechanism as a possible explanation for the partial agonist behavior of our spiroketal. Our co‐crystal structure, the first of a designed spiroketal–protein complex, suggests that spiroketals can be designed to selectively target other nuclear receptor subtypes.
Spiroketals for drug discovery: The structure‐based design, synthesis, and biochemical as well as structural validation of a spiroketal protein modulator are reported. The data suggests that the bisbenzannulated spiroketal functions as a potent partial agonist of the retinoid X receptor, thus highlighting the potential of spiroketals as de novo drug lead compounds.
Small ligands are a powerful way to control the function of protein complexes via dynamic binding interfaces. The classic example is found in gene transcription where small ligands regulate nuclear ...receptor binding to coactivator proteins via the dynamic activation function 2 (AF2) interface. Current ligands target the ligand‐binding pocket side of the AF2. Few ligands are known, which selectively target the coactivator side of the AF2, or which can be selectively switched from one side of the interface to the other. We use NMR spectroscopy and modeling to identify a natural product, which targets the retinoid X receptor (RXR) at both sides of the AF2. We then use chemical synthesis, cellular screening and X‐ray co‐crystallography to split this dual activity, leading to a potent and molecularly efficient RXR agonist, and a first‐of‐kind inhibitor selective for the RXR/coactivator interaction. Our findings justify future exploration of natural products at dynamic protein interfaces.
Rational splitting of the dual‐binding properties of a natural product delivers two mechanistically different ligands, which selectively target opposite sides of a dynamic protein interface (see picture; AF2=activation function 2). This work highlights the value of screening natural products against transient protein complexes.
Retinoid X receptors (RXRs) play key roles in many physiological processes in both the periphery and central nervous system. In addition, RXRs form heterodimers with other nuclear receptors to exert ...their physiological effects. The nuclear receptor related 1 protein (NURR1) is particularly interesting because of its role in promoting differentiation and survival of dopamine neurons. However, only a small number of RXR-heterodimer selective modulators are available, with limited chemical diversity. This work describes the synthesis, biochemical evaluation, and structural elucidation of a novel series of RXR ligands with strongly biased interactions with RXRα–NURR1 heterodimers. Targeted modifications to the small molecule biaryl scaffold caused local RXRα side-chain disturbances and displacement of secondary structural elements upon ligand binding. This resulted in the repositioning of protein helices in the heterodimer interface of RXRα, alterations in homo- versus heterodimer formation, and modulation of activation function 2 (AF2). The data provide a rationale for the design of RXR ligands consisting of a highly conserved hydrophilic region, strongly contributing to the ligand affinity, and a variable hydrophobic region, which efficiently probes the effects of structural changes at the level of the ligand on co-regulator recruitment or the RXRα–NURR1 dimerization interface.
We have previously described that diffuse large B-cell lymphomas (DLBCL) harboring an inflammatory and immunosuppressive microenvironment (i.e., IN-LME) carry independent poor prognosis while ...proof-of-principle experiments modifying the composition of the LME were sufficient to induce lymphoma regression in mice models ( Kotlov, Cancer Discovery 2021). However, identification of pharmaceutical approaches that by modifying the LME will induce an anti-lymphoma effect while being suitable for short-term human translation remains challenging. The IN-LME is characterized by the presence of pro-inflammatory and immunosuppressive chemokines and cytokines that frequently undergo post-translational modifications. QPCTL (glutaminyl-peptide cyclotransferase-like) is an intracellular enzyme that mediates a N-terminal pyroglutamylation of CD47 and several chemokines, including CCL2 and CCL7, ultimately resulting in their sustained expression and/or secretion. SC-2882 is a first-in-class specific QPCTL inhibitor that induces secondary proteolytic degradation of the monocyte chemoattractants CCL2 and CCL7 ( da Silva, Nature Immunology 2022) and inactivation of the “do-not-eat-me” signal CD47 ( Logtenberg Nature Medicine 2019). In a dataset of 470 DLBCL patients we found that the expression of QPCTL correlates negatively with overall survival and progression free survival (0.019 and <0.001, respectively). To determine whether QPCTL could be a potential target in DLBCL, we investigated its expression along with its targets CCL2, CCL7 and CD47 in additional 216 DLBCL cases categorized by LME. We found that while QPCTL and CD47 are expressed at similar levels in all DLBCL cases regardless their COO class and LME category, CCL2 was highly expressed in DLBCL harboring a IN-LME (p <0.05 vs. other LME categories), that also correlate with higher presence of tumor-associated macrophages (TAMs) and exhausted CD8+PD1 HIGH T cells (p <0.01 and p<0.001, vs. other LME categories, respectively). CCL7 was not expressed at detectable levels in majority of LMEs in DLBCL. In preclinical models, murine and human DLBCL cell lines expressed baseline QPCTL and CD47 while CCL2 and CCL7 expression (qPCR) and secretion (ELISA) increased at higher levels upon culturing these cells for 24 h embedded in splenic extracellular matrix 3D cultures. Exposure of a panel of 3 murine and 5 human DLBCL cells to increasing concentrations of SC-2882 (vs. vehicle) for up to 72 h and up to a 10 micromolar dose had no noticeable decrease in cell viability or proliferation. However, SC-2882 (vs. vehicle) at nanomolar concentrations increased anti-Cd20-dependent lymphoma cell phagocytosis by in vitro differentiated macrophages (from splenic monocytes in mice and THP1 cells in humans) in 3D co-cultures (p < 0.05, for all conditions). To further test the anti-lymphoma effect of SC-2882 we used a murine GCB-DLBCL model by implanting GEMM-derived aggressive Ezh2 mutant (EZH2 Y646equivalent) + BCL2 amplified lymphoma cells in the spleen of immunocompetent C57BL/mice (n=14). After implantation, mice were randomized to receive SC-2882 by oral gavage (n=7) or vehicle (n=14) for 14 days. Tumor burden was followed by luciferase imaging (Caliper), and spleens were analyzed at sacrifice by multiparametric imaging, flow-cytometry, and spatial transcriptomics. Oral SC-2882 (vs. vehicle) induced a significant decrease in tumor growth (p=0.0079, Figure 1) without evidence of systemic toxicity by body weight, blood cell count and chemistry panel. Analysis of the LME in remnant splenic tumors showed in SC-2882 (vs. vehicle) treated mice a significant decrease in lymphoma cells and Ki67+ cells (p<0.001) as well as F480+ TAMs (p<0.01) while there was an increase of CD3+ T cell infiltration (p<0.001). We also found a decrease in Cd4+Foxp3+ Tregs and increase in Cd8+Gzb+ cells in SC-2882 treated mice by flow-cytometry. These changes mirrored the findings in the spatial transcriptomics. Additional analysis of LME cell subpopulations by SC-RNA-seq is ongoing. Overall, these data indicate that QPCTL is a potential target in DLBCL, and that QPCTL inhibition exhibits potent anti-lymphoma effects primarily by targeting the IN-LME.
Abstract Background: SC-2882 is an orally administered first-in-class small molecule inhibitor of glutaminyl-peptide cyclotransferase like (QPCTL). QPCTL is an intracellular enzyme that modifies ...several proteins containing an N-terminal glutamine or glutamate, resulting in pyroglutamylated amino acids. Pyroglutamylation can alter protein function in different ways and dependent on context. Key substrate proteins include the “don’t eat me” signal protein CD47 and several chemokines (CCL2 and CCL7) (Nat Med 2019, Nat Imm. 2022). Previously, we have shown activity of SC-2882 in a murine model of diffuse large B-cell lymphoma (Di Siervi, 2023). Here we report for the first time, the evaluation of SC-2882 in several preclinical mouse models for solid tumors. Methods: Potency of SC-2882 was evaluated in a cell-based assay in several human and mouse tumor cell lines: SIRPα binding to CD47 in response to increasing concentrations of SC-2882 was measured in a flowcytometry-based assay. Several syngeneic mouse tumor models were evaluated for their response towards SC-2882 as a single agent and in combination with either a murine anti-PD-L1 antibody or cisplatin. Animals were dosed orally with SC-2882 once daily for the duration of the experiment. Results: In all human and mouse cancer cell lines tested, SC-2882 was able to reduce SIRPα binding to CD47 significantly. This reduction was equivalent to the reduction observed in QPCTL genetic knockouts created in the same cell line. In the same experiments, no differences in CD47 expression were observed. From anti-tumor efficacy studies, we learned that SC-2882 was well tolerated in mice as a single agent and in combination with anti-PD-L1 or cisplatin for at least 4 weeks (this was the maximum period tested). No clinical signs or changes in body weight were observed, even over prolonged periods of treatment. Tumor growth inhibition and tumor shrinkage by SC-2882 was observed in 5 different syngeneic mouse tumor models. In two out of these five models, SC-2882 was able to reduce tumor growth as a single agent. In the other three models, tumor growth inhibition was observed in combination with either anti-PD-L1 or cisplatin. Conclusions: SC-2882 is an orally administered small molecule inhibitor with a favorable PK profile. Preclinical studies show that SC-2882 was well tolerated in mice. In several mouse tumor models, SC-2882 reduced tumor growth in either single agent treated animals or in combination with anti-PD-L1 or cisplatin. SC-2882 is currently in IND enabling studies and scheduled to enter the clinic in 2024. Citation Format: Jasper Mullenders, Marcel Scheepstra, Michel R. Faas, Annette B. Galler, Bastiaan Evers, Jens U. Wuerthner. SC-2882, a novel QPCTL inhibitor, with anti-tumor activity in solid tumor models abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4690.
Retinoic acid receptor-related orphan receptor γt (RORγt) is a nuclear receptor associated with the pathogenesis of autoimmune diseases. Allosteric inhibition of RORγt is conceptually new, unique for ...this specific nuclear receptor, and offers advantages over traditional orthosteric inhibition. Here, we report a highly efficient in silico-guided approach that led to the discovery of novel allosteric RORγt inverse agonists with a distinct isoxazole chemotype. The the most potent compound, 25 (FM26), displayed submicromolar inhibition in a coactivator recruitment assay and effectively reduced IL-17a mRNA production in EL4 cells, a marker of RORγt activity. The projected allosteric mode of action of 25 was confirmed by biochemical experiments and cocrystallization with the RORγt ligand binding domain. The isoxazole compounds have promising pharmacokinetic properties comparable to other allosteric ligands but with a more diverse chemotype. The efficient ligand-based design approach adopted demonstrates its versatility in generating chemical diversity for allosteric targeting of RORγt.
RORγt is critical for the differentiation and proliferation of Th17 cells associated with several chronic autoimmune diseases. We report the discovery of a novel allosteric binding site on the ...nuclear receptor RORγt. Co-crystallization of the ligand binding domain (LBD) of RORγt with a series of small-molecule antagonists demonstrates occupancy of a previously unreported allosteric binding pocket. Binding at this non-canonical site induces an unprecedented conformational reorientation of helix 12 in the RORγt LBD, which blocks cofactor binding. The functional consequence of this allosteric ligand-mediated conformation is inhibition of function as evidenced by both biochemical and cellular studies. RORγt function is thus antagonized in a manner molecularly distinct from that of previously described orthosteric RORγt ligands. This brings forward an approach to target RORγt for the treatment of Th17-mediated autoimmune diseases. The elucidation of an unprecedented modality of pharmacological antagonism establishes a mechanism for modulation of nuclear receptors.
Evasion of apoptosis is critical for the development and growth of tumors. The pro-survival protein myeloid cell leukemia 1 (Mcl-1) is an antiapoptotic member of the Bcl-2 family, associated with ...tumor aggressiveness, poor survival, and drug resistance. Development of Mcl-1 inhibitors implies blocking of protein–protein interactions, generally requiring a lengthy optimization process of large, complex molecules. Herein, we describe the use of DNA-encoded chemical library synthesis and screening to directly generate complex, yet conformationally privileged macrocyclic hits that serve as Mcl-1 inhibitors. By applying a conceptual combination of conformational analysis and structure-based design in combination with a robust synthetic platform allowing rapid analoging, we optimized in vitro potency of a lead series into the low nanomolar regime. Additionally, we demonstrate fine-tuning of the physicochemical properties of the macrocyclic compounds, resulting in the identification of lead candidates 57/59 with a balanced profile, which are suitable for future development toward therapeutic use.
Cucurbit8uril is a supramolecular inducer of protein heterodimerization for proteins appended with methylviologen and naphthalene host elements. Two sets of fluorescent protein pairs, which visualize ...the specific protein assembly process, enabled the interplay of the supramolecular elements with the proteins to be established.
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