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Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules.

ABSTRACT: Chemical biology strategies for directly perturbing protein homeostasis including the degradation tag (dTAG) system provide temporal advantages over genetic approaches and improved selectivity over small molecule inhibitors. We describe dTAGV-1, an exclusively selective VHL-recruiting dTAG molecule, to rapidly degrade FKBP12F36V-tagged proteins. dTAGV-1 overcomes a limitation of previously reported CRBN-recruiting dTAG molecules to degrade recalcitrant oncogenes, supports combination degrader studies and facilitates investigations of protein function in cells and mice.

SUBMITTER: Nabet B

PROVIDER: S-EPMC7501296 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules.

Nabet Behnam B Ferguson Fleur M FM Seong Bo Kyung A BKA Kuljanin Miljan M Leggett Alan L AL Mohardt Mikaela L ML Robichaud Amanda A Conway Amy S AS Buckley Dennis L DL Mancias Joseph D JD Bradner James E JE Stegmaier Kimberly K Gray Nathanael S NS

Nature communications 20200918 1

Chemical biology strategies for directly perturbing protein homeostasis including the degradation tag (dTAG) system provide temporal advantages over genetic approaches and improved selectivity over small molecule inhibitors. We describe dTAGV-1, an exclusively selective VHL-recruiting dTAG molecule, to rapidly degrade FKBP12F36V-tagged proteins. dTAGV-1 overcomes a limitation of previously reported CRBN-recruiting dTAG molecules to degrade recalcitrant oncogenes, ...[more]

PMID: 32948771

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Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules

Project description:Chemical biology strategies for directly perturbing protein homeostasis including the degradation tag (dTAG) system provide temporal advantages over genetic approaches and improved selectivity over small molecule inhibitors. We describe dTAGV-1, an exclusively selective VHL-recruiting dTAG molecule, to rapidly degrade FKBP12F36V-tagged proteins. dTAGV-1 overcomes a limitation of previously reported CRBN-recruiting dTAG molecules to degrade recalcitrant oncogenes, supports combination degrader studies and facilitates investigations of protein function in cells and mice

2020-09-21 | PXD018937 | Pride

Chemical control over immune recognition: a class of antibody-recruiting small molecules that target prostate cancer.

Project description:Prostate cancer is the second leading cause of cancer-related death among the American male population, and society is in dire need of new approaches to treat this disease. Here we report the design, synthesis, and biological evaluation of a class of bifunctional small molecules called antibody-recruiting molecules targeting prostate cancer (ARM-Ps) that enhance the recognition of prostate cancer cells by the human immune system. ARM-P derivatives were designed rationally via the computational analysis of crystallographic data, and we demonstrate here that these materials are able to (1) bind prostate-specific membrane antigen (PSMA) with high affinity (high pM to low nM), (2) template the formation of ternary complexes of anti-DNP antibodies, ARM-P, and LNCaP human prostate cancer cells, and (3) mediate the antibody-dependent killing of LNCaP cells in the presence of human effector cells. This manuscript describes the application of fundamental chemical principles to the design of a novel class of molecules with high therapeutic potential. We believe that this general small-molecule-based strategy could give rise to novel directions in treating cancer and other diseases.

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Targeted Degradation of Oncogenic KRASG12C by VHL-Recruiting PROTACs.

Project description:KRAS is mutated in ?20% of human cancers and is one of the most sought-after targets for pharmacological modulation, despite having historically been considered "undruggable." The discovery of potent covalent inhibitors of the KRASG12C mutant in recent years has sparked a new wave of interest in small molecules targeting KRAS. While these inhibitors have shown promise in the clinic, we wanted to explore PROTAC-mediated degradation as a complementary strategy to modulate mutant KRAS. Herein, we report the development of LC-2, the first PROTAC capable of degrading endogenous KRASG12C. LC-2 covalently binds KRASG12C with a MRTX849 warhead and recruits the E3 ligase VHL, inducing rapid and sustained KRASG12C degradation leading to suppression of MAPK signaling in both homozygous and heterozygous KRASG12C cell lines. LC-2 demonstrates that PROTAC-mediated degradation is a viable option for attenuating oncogenic KRAS levels and downstream signaling in cancer cells.

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Von Hippel-Lindau (VHL) small-molecule inhibitor binding increases stability and intracellular levels of VHL protein.

Project description:Von Hippel-Lindau (VHL) disease is characterized by frequent mutation of VHL protein, a tumor suppressor that functions as the substrate recognition subunit of a Cullin2 RING E3 ligase complex (CRL2VHL). CRL2VHL plays important roles in oxygen sensing by targeting hypoxia-inducible factor-alpha (HIF-α) subunits for ubiquitination and degradation. VHL is also commonly hijacked by bifunctional molecules such as proteolysis-targeting chimeras to induce degradation of target molecules. We previously reported the design and characterization of VHL inhibitors VH032 and VH298 that block the VHL:HIF-α interaction, activate the HIF transcription factor, and induce a hypoxic response, which can be beneficial to treat anemia and mitochondrial diseases. How these compounds affect the global cellular proteome remains unknown. Here, we use unbiased quantitative MS to identify the proteomic changes elicited by the VHL inhibitor compared with hypoxia or the broad-spectrum prolyl-hydroxylase domain enzyme inhibitor IOX2. Our results demonstrate that VHL inhibitors selectively activate the HIF response similar to the changes induced in hypoxia and IOX2 treatment. Interestingly, VHL inhibitors were found to specifically upregulate VHL itself. Our analysis revealed that this occurs via protein stabilization of VHL isoforms and not via changes in transcript levels. Increased VHL levels upon VH298 treatment resulted in turn in reduced levels of HIF-1α protein. This work demonstrates the specificity of VHL inhibitors and reveals different antagonistic effects upon their acute versus prolonged treatment in cells. These findings suggest that therapeutic use of VHL inhibitors may not produce overt side effects from HIF stabilization as previously thought.

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A selective and orally bioavailable VHL-recruiting PROTAC achieves SMARCA2 degradation in vivo.

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Direct transcriptional control of the Dpp target omb by the DNA binding protein Brinker.

Project description:The gradient morphogen Decapentaplegic (Dpp) organizes pattern by inducing the transcription of different target genes at distinct threshold concentrations during Drosophila development. An important, albeit indirect, mode by which Dpp controls the spatial extent of its targets is via the graded downregulation of brinker, whose product in turn negatively regulates the expression of these targets. Here we report the molecular dissection of the cis-regulatory sequences of optomotor-blind (omb), a Dpp target gene in the wing. We identify a minimal 284 bp Dpp response element and demonstrate that it is subject to Brinker (Brk) repression. Using this omb wing enhancer, we show that Brk is a sequence-specific DNA binding protein. Mutations in the high-affinity Brk binding site abolish responsiveness of this omb enhancer to Brk and also compromise the input of an unknown transcriptional activator. Our results therefore identify Brk as a novel transcription factor antagonizing Dpp signalling by directly binding target genes and repressing their expression.

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Anti-proliferative protein Tob negatively regulates CPEB3 target by recruiting Caf1 deadenylase.

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Identification of direct protein targets of small molecules.

Project description:Small-molecule target identification is a vital and daunting task for the chemical biology community as well as for researchers interested in applying the power of chemical genetics to impact biology and medicine. To overcome this "target ID" bottleneck, new technologies are being developed that analyze protein-drug interactions, such as drug affinity responsive target stability (DARTS), which aims to discover the direct binding targets (and off targets) of small molecules on a proteome scale without requiring chemical modification of the compound. Here, we review the DARTS method, discuss why it works, and provide new perspectives for future development in this area.

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Development of Selective Histone Deacetylase 6 (HDAC6) Degraders Recruiting Von Hippel-Lindau (VHL) E3 Ubiquitin Ligase.

Project description:Histone deacetylase 6 (HDAC6) is involved in multiple cellular processes such as aggresome formation, protein stability, and cell motility. Numerous HDAC6-selective inhibitors have been developed as cellular chemical tools to elucidate the function of HDAC6. Since HDAC6 has multiple domains that cannot be studied by HDAC6-selective inhibitors, CRISPR-CAS9 and siRNA/shRNA have been employed to elucidate the nonenzymatic functions of HDAC6. However, these genetic methods have many limitations. Proteolysis targeting chimera (PROTAC) is an emerging technology for the development of small molecules that can quickly remove the entire protein in cells. We previously developed multifunctional HDAC6 degraders that can recruit cereblon (CRBN) E3 ubiquitin ligase. These HDAC6 degraders can degrade not only HDAC6 but also neo-substrates of CRBN. They are excellent candidates for the development of anticancer therapeutics, but the multifunctional nature of the CRBN-based HDAC6 degraders has limited their utility as specific chemical probes for the study of HDAC6-related cellular pathways. Herein we report the development of the first cell-permeable HDAC6-selective degraders employing Von Hippel-Lindau (VHL) E3 ubiquitin ligase, which does not have any known neo-substrates. The DC50's of the most potent compound 3j are 7.1 nM and 4.3 nM in human MM1S and mouse 4935 cell lines, respectively. The D max's of 3j in these two cell lines are 90% and 57%, respectively.

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Project description:Heterotrimeric guanine nucleotide-binding proteins (G proteins) composed of three subunits ?, ?, ? mediate activation of multiple intracellular signaling cascades initiated by G protein-coupled receptors (GPCRs). Previously our laboratory identified small molecules that bind to G?? and interfere with or enhance binding of select effectors with G??. To understand the molecular mechanisms of selectivity and assess binding of compounds to G??, we used biophysical and biochemical approaches to directly monitor small molecule binding to G??. Surface plasmon resonance (SPR) analysis indicated that multiple compounds bound directly to G?? with affinities in the high nanomolar to low micromolar range but with surprisingly slow on and off rate kinetics. While the k(off) was slow for most of the compounds in physiological buffers, they could be removed from G?? with mild chaotropic salts or mildly dissociating collision energy in a mass-spectrometer indicating that compound-G?? interactions were non-covalent. Finally, at concentrations used to observe maximal biological effects the stoichiometry of binding was 1:1. The results from this study show that small molecule modulation of G??-effector interactions is by specific direct non-covalent and reversible binding of small molecules to G??. This is highly relevant to development of G?? targeting as a therapeutic approach since reversible, direct binding is a prerequisite for drug development and important for specificity.

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